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1.  Proceedings of the 14th annual conference of INEBRIA 
Holloway, Aisha S. | Ferguson, Jennifer | Landale, Sarah | Cariola, Laura | Newbury-Birch, Dorothy | Flynn, Amy | Knight, John R. | Sherritt, Lon | Harris, Sion K. | O’Donnell, Amy J. | Kaner, Eileen | Hanratty, Barbara | Loree, Amy M. | Yonkers, Kimberly A. | Ondersma, Steven J. | Gilstead-Hayden, Kate | Martino, Steve | Adam, Angeline | Schwartz, Robert P. | Wu, Li-Tzy | Subramaniam, Geetha | Sharma, Gaurav | McNeely, Jennifer | Berman, Anne H. | Kolaas, Karoline | Petersén, Elisabeth | Bendtsen, Preben | Hedman, Erik | Linderoth, Catharina | Müssener, Ulrika | Sinadinovic, Kristina | Spak, Fredrik | Gremyr, Ida | Thurang, Anna | Mitchell, Ann M. | Finnell, Deborah | Savage, Christine L. | Mahmoud, Khadejah F. | Riordan, Benjamin C. | Conner, Tamlin S. | Flett, Jayde A. M. | Scarf, Damian | McRee, Bonnie | Vendetti, Janice | Gallucci, Karen Steinberg | Robaina, Kate | Clark, Brendan J. | Jones, Jacqueline | Reed, Kathryne D. | Hodapp, Rachel M. | Douglas, Ivor | Burnham, Ellen L. | Aagaard, Laura | Cook, Paul F. | Harris, Brett R. | Yu, Jiang | Wolff, Margaret | Rogers, Meighan | Barbosa, Carolina | Wedehase, Brendan J. | Dunlap, Laura J. | Mitchell, Shannon G. | Dusek, Kristi A. | Gryczynski, Jan | Kirk, Arethusa S. | Oros, Marla T. | Hosler, Colleen | O’Grady, Kevin E. | Brown, Barry S. | Angus, Colin | Sherborne, Sidney | Gillespie, Duncan | Meier, Petra | Brennan, Alan | de Vargas, Divane | Soares, Janaina | Castelblanco, Donna | Doran, Kelly M. | Wittman, Ian | Shelley, Donna | Rotrosen, John | Gelberg, Lillian | Edelman, E. Jennifer | Maisto, Stephen A. | Hansen, Nathan B. | Cutter, Christopher J. | Deng, Yanhong | Dziura, James | Fiellin, Lynn E. | O’Connor, Patrick G. | Bedimo, Roger | Gibert, Cynthia | Marconi, Vincent C. | Rimland, David | Rodriguez-Barradas, Maria C. | Simberkoff, Michael S. | Justice, Amy C. | Bryant, Kendall J. | Fiellin, David A. | Giles, Emma L. | Coulton, Simon | Deluca, Paolo | Drummond, Colin | Howel, Denise | McColl, Elaine | McGovern, Ruth | Scott, Stephanie | Stamp, Elaine | Sumnall, Harry | Vale, Luke | Alabani, Viviana | Atkinson, Amanda | Boniface, Sadie | Frankham, Jo | Gilvarry, Eilish | Hendrie, Nadine | Howe, Nicola | McGeechan, Grant J. | Ramsey, Amy | Stanley, Grant | Clephane, Justine | Gardiner, David | Holmes, John | Martin, Neil | Shevills, Colin | Soutar, Melanie | Chi, Felicia W. | Weisner, Constance | Ross, Thekla B. | Mertens, Jennifer | Sterling, Stacy A. | Shorter, Gillian W. | Heather, Nick | Bray, Jeremy | Cohen, Hildie A. | McPherson, Tracy L. | Adam, Cyrille | López-Pelayo, Hugo | Gual, Antoni | Segura-Garcia, Lidia | Colom, Joan | Ornelas, India J. | Doyle, Suzanne | Donovan, Dennis | Duran, Bonnie | Torres, Vanessa | Gaume, Jacques | Grazioli, Véronique | Fortini, Cristiana | Paroz, Sophie | Bertholet, Nicolas | Daeppen, Jean-Bernard | Satterfield, Jason M. | Gregorich, Steven | Alvarado, Nicholas J. | Muñoz, Ricardo | Kulieva, Gozel | Vijayaraghavan, Maya | Adam, Angéline | Cunningham, John A. | Díaz, Estela | Palacio-Vieira, Jorge | Godinho, Alexandra | Kushir, Vladyslav | O’Brien, Kimberly H. M. | Aguinaldo, Laika D. | Sellers, Christina M. | Spirito, Anthony | Chang, Grace | Blake-Lamb, Tiffany | LaFave, Lea R. Ayers | Thies, Kathleen M. | Pepin, Amy L. | Sprangers, Kara E. | Bradley, Martha | Jorgensen, Shasta | Catano, Nico A. | Murray, Adelaide R. | Schachter, Deborah | Andersen, Ronald M. | Rey, Guillermina Natera | Vahidi, Mani | Rico, Melvin W. | Baumeister, Sebastian E. | Johansson, Magnus | Sinadinovic, Christina | Hermansson, Ulric | Andreasson, Sven | O’Grady, Megan A. | Kapoor, Sandeep | Akkari, Cherine | Bernal, Camila | Pappacena, Kristen | Morley, Jeanne | Auerbach, Mark | Neighbors, Charles J. | Kwon, Nancy | Conigliaro, Joseph | Morgenstern, Jon | Magill, Molly | Apodaca, Timothy R. | Borsari, Brian | Hoadley, Ariel | Scott Tonigan, J. | Moyers, Theresa | Fitzgerald, Niamh M. | Schölin, Lisa | Barticevic, Nicolas | Zuzulich, Soledad | Poblete, Fernando | Norambuena, Pablo | Sacco, Paul | Ting, Laura | Beaulieu, Michele | Wallace, Paul George | Andrews, Matthew | Daley, Kate | Shenker, Don | Gallagher, Louise | Watson, Rod | Weaver, Tim | Bruguera, Pol | Oliveras, Clara | Gavotti, Carolina | Barrio, Pablo | Braddick, Fleur | Miquel, Laia | Suárez, Montse | Bruguera, Carla | Brown, Richard L. | Capell, Julie Whelan | Paul Moberg, D. | Maslowsky, Julie | Saunders, Laura A. | McCormack, Ryan P. | Scheidell, Joy | Gonzalez, Mirelis | Bauroth, Sabrina | Liu, Weiwei | Lindsay, Dawn L. | Lincoln, Piper | Hagle, Holly | Wallhed Finn, Sara | Hammarberg, Anders | Andréasson, Sven | King, Sarah E. | Vargo, Rachael | Kameg, Brayden N. | Acquavita, Shauna P. | Van Loon, Ruth Anne | Smith, Rachel | Brehm, Bonnie J. | Diers, Tiffiny | Kim, Karissa | Barker, Andrea | Jones, Ashley L. | Skinner, Asheley C. | Hinman, Agatha | Svikis, Dace S. | Thacker, Casey L. | Resnicow, Ken | Beatty, Jessica R. | Janisse, James | Puder, Karoline | Bakshi, Ann-Sofie | Milward, Joanna M. | Kimergard, Andreas | Garnett, Claire V. | Crane, David | Brown, Jamie | West, Robert | Michie, Susan | Rosendahl, Ingvar | Andersson, Claes | Gajecki, Mikael | Blankers, Matthijs | Donoghue, Kim | Lynch, Ellen | Maconochie, Ian | Phillips, Ceri | Pockett, Rhys | Phillips, Tom | Patton, R. | Russell, Ian | Strang, John | Stewart, Maureen T. | Quinn, Amity E. | Brolin, Mary | Evans, Brooke | Horgan, Constance M. | Liu, Junqing | McCree, Fern | Kanovsky, Doug | Oberlander, Tyler | Zhang, Huan | Hamlin, Ben | Saunders, Robert | Barton, Mary B. | Scholle, Sarah H. | Santora, Patricia | Bhatt, Chirag | Ahmed, Kazi | Hodgkin, Dominic | Gao, Wenwu | Merrick, Elizabeth L. | Drebing, Charles E. | Larson, Mary Jo | Sharma, Monica | Petry, Nancy M. | Saitz, Richard | Weisner, Constance M. | Young-Wolff, Kelly C. | Lu, Wendy Y. | Blosnich, John R. | Lehavot, Keren | Glass, Joseph E. | Williams, Emily C. | Bensley, Kara M. | Chan, Gary | Dombrowski, Julie | Fortney, John | Rubinsky, Anna D. | Lapham, Gwen T. | Forray, Ariadna | Olmstead, Todd A. | Gilstad-Hayden, Kathryn | Kershaw, Trace | Dillon, Pamela | Weaver, Michael F. | Grekin, Emily R. | Ellis, Jennifer D. | McGoron, Lucy | McGoron, Lucy
doi:10.1186/s13722-017-0087-8
PMCID: PMC5606215
2.  Evidence for an Induced Conformational Change in the Catalytic Mechanism of Homoisocitrate Dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N Mutant Enzyme 
Homoisocitrate dehydrogenase (HIcDH) catalyzes the NAD+-dependent oxidative decarboxylation of HIc to α-ketoadipate, the fourth step in the α-aminoadipate pathway responsible for the de novo synthesis of L-lysine in fungi. A mechanism has been proposed for the enzyme that makes use of a Lys-Tyr pair as acid-base catalysts, with Lys acting as a base to accept a proton from the α-hydroxyl of homoisocitrate, and Tyr acting as an acid to protonate the C3 of the enol of α-ketoadipate in the enolization reaction. Three conserved aspartate residues, D243, D267 and D271, coordinate Mg2+, which is also coordinated to the α-carboxylate and α-hydroxyl of homoisocitrate. On the basis of kinetic isotope effects, it was proposed that a conformational change to close the active site and organize the active site for catalysis contributed to rate limitation of the overall reaction of the Saccharomyces cerevisiae HIcDH (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., and Cook, P. F. (2008) Biochemistry 47, 4169–4180.). In order to test this hypothesis, site-directed mutagenesis was used to change D271, a metal ion ligand and binding determinant for MgHIc, to N. The mutant enzyme was characterized using initial rate studies. A decrease of 520-fold was observed in V and V/KMgHIc, suggesting the same step(s) limit the reaction at limiting and saturating MgHIc concentrations. Solvent kinetic deuterium isotope effects (SKIE) and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations. In addition, at limiting MgHIc, an inverse (SKIE) of 0.7 coupled to a significant normal effect of viscosogen (2.1) indicates equilibrium binding of MgHIc prior to the rate-limiting conformational change. The maximum rate exhibits a small partial change at high pH suggesting a pH-dependent conformational change, while V/KMgHIc exhibits the same partial change observed in V, and a decrease at low pH with a pKa of 6 reflecting the requirement for the unprotonated form of MgHIc to bind to enzyme. However, neither parameter reflects the pH dependence of the chemical reaction. This pH independence of the chemical reaction over the range 5.5–9.5 is consistent with the much slower conformational change that would effectively perturb the observed pK values for catalytic groups to lower and higher pH. In other words, the pH dependence of the chemical reaction will only be observed when chemistry becomes slower than the rate of the conformational change. Data support the hypothesis of the existence of a pre-catalytic conformational change coupled to the binding of MgHIc.
doi:10.1016/j.abb.2015.08.016
PMCID: PMC4587348  PMID: 26325079
3.  Probing the Chemical Mechanism of Saccharopine Reductase from Saccharomyces cerevisiae Using Site-Directed Mutagenesis 
Saccharopine reductase catalyzes the reductive amination of L-α-aminoadipate-δ-semialdehyde with L-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid-base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid-base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue.
doi:10.1016/j.abb.2015.08.023
PMCID: PMC4596410  PMID: 26342457
Saccharopine Reductase; site-directed mutagenesis; initial rate studies; pH-rate profiles; isotope effects; viscosity
4.  A counselor in your pocket: feasibility of mobile health tailored messages to support HIV medication adherence 
Purpose
Medication adherence is a major challenge in HIV treatment. New mobile technologies such as smartphones facilitate the delivery of brief tailored messages to promote adherence. However, the best approach for tailoring messages is unknown. Persons living with HIV (PLWH) might be more receptive to some messages than others based on their current psychological state.
Methods
We recruited 37 PLWH from a parent study of motivational states and adherence. Participants completed smartphone-based surveys at a random time every day for 2 weeks, then immediately received intervention or control tailored messages, depending on random assignment. After 2 weeks in the initial condition, participants received the other condition in a crossover design. Intervention messages were tailored to match PLWH’s current psychological state based on five variables – control beliefs, mood, stress, coping, and social support. Control messages were tailored to create a mismatch between message framing and participants’ current psychological state. We evaluated intervention feasibility based on acceptance, ease of use, and usefulness measures. We also used pilot randomized controlled trial methods to test the intervention’s effect on adherence, which was measured using electronic caps that recorded pill-bottle openings.
Results
Acceptance was high based on 76% enrollment and 85% satisfaction. Participants found the hardware and software easy to use. However, attrition was high at 59%, and usefulness ratings were slightly lower. The most common complaint was boredom. Unexpectedly, there was no difference between mismatched and matched messages’ effects, but each group showed a 10%–15% improvement in adherence after crossing to the opposite study condition.
Conclusion
Although smartphone-based tailored messaging was feasible and participants had clinically meaningful improvements in adherence, the mechanisms of change require further study. Possible explanations might include novelty effects, increased receptiveness to new information after habituation, or pseudotailoring, three ways in which attentional processes can affect behavior.
Video abstract
doi:10.2147/PPA.S88222
PMCID: PMC4599065  PMID: 26491263
adherence; communication; feasibility; HIV; technology
5.  Predictors of Adherence to Glaucoma Treatment in a Multi-Site Study 
Background
Poor adherence hinders glaucoma treatment. Studies have identified demographic and clinical predictors of adherence but fewer psychological variables.
Purpose
We examined predictors from four health behavior theories and past research.
Methods
In the baseline phase of a 3-site adherence study, before any intervention, 201 participants used electronic Medication Event Monitoring System (MEMS) bottles to monitor eyedrop use for 2 months, and completed questionnaires including self-reported adherence.
Results
MEMS showed 79% adherence and self-report 94% (0.5–1.5 missed weekly doses), but correlated only rs = .31. Self-efficacy, motivation, dose frequency, and non-minority race/ethnicity predicted 35% of variance in MEMS. Cues to action, self-efficacy, and intention predicted 20% of variance in self-reported adherence.
Conclusions
Self-efficacy, motivation, intention, cues to action, dose frequency, and race/ethnicity each independently predicted adherence. Other predictors from all theories were supported in bivariate analyses, but additional study is needed. Researchers and clinicians should consider psychological predictors of adherence.
doi:10.1007/s12160-014-9641-8
PMCID: PMC4336606  PMID: 25248302
attitude; glaucoma; medication adherence; motivation; theory
6.  Brief report on ecological momentary assessment: everyday states predict HIV prevention behaviors 
BMC Research Notes  2016;9:9.
Background
Prevention behaviors help persons living with HIV (PLWH) to avoid transmitting HIV, and psychological variables have been found to predict HIV prevention behaviors. These variables have typically been measured using retrospective questionnaires about average psychological states over a period of time, which are likely to be biased by selective recall and interpretation. Measuring the same variables as momentary states, in the day-to-day context where they actually occur, may reveal different relationships to behavior.
Findings
21 PLWH completed daily surveys about momentary states and prevention behaviors. Brief, validated measures were used to assess control beliefs, mood, stress, coping, social support, stigma, knowledge, and motivation. We used multilevel models to predict prevention behaviors from momentary states the previous day, while controlling for the effect of multiple observations from the same person over time. Participants reported a moderate overall level of HIV prevention behaviors during the 6-month study. Although lapses in prevention were infrequent, there was room for improvement. Control beliefs, mood, and motivation had significant prospective effects on HIV prevention behaviors, rs = 0.07−0.21. Stress and coping had effects approaching significance.
Conclusions
Some momentary states predicted prevention behaviors, providing partial support for the motivational model. This finding supports past research showing effects of momentary states on behavior, and advances the science by testing multiple predictors. High within-sample diversity strengthened generalizability, but the overall sample size was small and the findings require replication. Future research should continue to examine the everyday experiences of PLWH as influences on their behavior.
doi:10.1186/s13104-015-1814-4
PMCID: PMC4700569  PMID: 26728848
Ecological momentary assessment; HIV; Motivation; Prevention; Theory
7.  A Novel Quantitative Approach to Concept Analysis: The Internomological Network 
Nursing research  2012;61(5):369-378.
Background
When a construct such as patients’ transition to self-management of chronic illness is studied by researchers across multiple disciplines, the meaning of key terms can become confused. This results from inherent problems in language where a term can have multiple meanings (polysemy) and different words can mean the same thing (synonymy).
Objectives
To test a novel quantitative method for clarifying the meaning of constructs by examining the similarity of published contexts in which they are used.
Method
Published terms related to the concept transition to self-management of chronic illness were analyzed using the internomological network (INN), a type of latent semantic analysis to calculate the mathematical relationships between constructs based on the contexts in which researchers use each term. This novel approach was tested by comparing results to those from concept analysis, a best-practice qualitative approach to clarifying meanings of terms. By comparing results of the two methods, the best synonyms of transition to self-management, as well as key antecedent, attribute, and consequence terms, were identified.
Results
Results from INN analysis were consistent with those from concept analysis. The potential synonyms self-management, transition, and adaptation had the greatest utility. Adaptation was the clearest overall synonym, but had lower cross-disciplinary use. The terms coping and readiness had more circumscribed meanings. The INN analysis confirmed key features of transition to self-management, and suggested related concepts not found by the previous review.
Discussion
The INN analysis is a promising novel methodology that allows researchers to quantify the semantic relationships between constructs. The method works across disciplinary boundaries, and may help to integrate the diverse literature on self-management of chronic illness.
doi:10.1097/NNR.0b013e318250c199
PMCID: PMC3422604  PMID: 22592387
chronic disease; concept analysis; construct validity; factor analysis; self-management
8.  Contribution of K99 and D319 to Substrate Binding and Catalysis in the Saccharopine Dehydrogenase Reaction 
Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to L-lysine and α-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the α-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V2/Km values for lysine, α-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/KLys increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V2/KNADH by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and α-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH.
doi:10.1016/j.abb.2011.07.013
PMCID: PMC3174770  PMID: 21819960
9.  Patterns of Change in Symptom Clusters with HIV Disease Progression 
Context
With better antiretroviral treatments (ART), persons living with HIV (PLWH) are living longer, healthier lives. Therefore, they also experience more medical comorbidities that come with normal aging, as well as side effects of multiple treatments and long-term sequelae of HIV. It can be hard to know whether symptoms reported by PLWH are related to comorbidities or are signs of HIV disease progression and possible treatment failure.
Objectives
The current study was designed to disentangle these issues by examining within-person symptom changes in data collected from a cohort of PLWH before the advent of highly efficacious ART.
Methods
This study was a secondary analysis of symptom reports in longitudinal data collected from 246 PLWH in 1992–1994. Multilevel modeling was used to test for changes over time in HIV-related symptom clusters. Analyses also tested the effects of person-level demographic covariates and co-occurring mental health symptoms on HIV symptoms, and examined the magnitude of within-person versus between-person variations in reported symptom severity.
Results
Two of six HIV-related symptom clusters, malaise/fatigue and nausea/vomiting, increased over time in the context of HIV disease progression, while the other four did not. Changes were independent of baseline disease severity or psychological covariates. There was substantial within-person variability in absolute symptom severity.
Conclusion
Relatively small but consistent changes in symptoms related to nausea or fatigue may suggest HIV disease progression, while changes in other HIV symptom clusters may instead be related to comorbidities or normal aging. Further research is recommended on symptom progression in PLWH.
doi:10.1016/j.jpainsymman.2010.09.021
PMCID: PMC3132274  PMID: 21429701
Aging; fatigue; HIV; nausea; symptom clusters
10.  Anxiety, Depression, Stress, and Cortisol Levels in Mothers of Children Undergoing Maintenance Therapy for Childhood Acute Lymphoblastic Leukemia 
The purpose of this study was to compare anxiety, depression, and stress between mothers of children during maintenance treatment for acute lymphoblastic leukemia (ALL) and matched controls. Twenty-six mothers were recruited from the hematology unit at a children’s hospital, and 26 mothers were recruited from the community. Participants were matched to their child’s age and gender. Mothers completed the Hospital Anxiety and Depression Scale, the Perceived Stress Sale, and collected salivary cortisol 4 times a day for 3 consecutive days. Compared with mothers of healthy children, anxiety scores did not differ (P = .10), but depression scores were higher (P = .003) in mothers of children with ALL. More mothers in the ALL group scored above the cutoff of 7 indicating clinical anxiety (46%) and depressive symptoms (27%). A trend toward increased stress was found in mothers in the ALL group. No difference was found in overall daily cortisol (area under the curve), daily decrease in cortisol (slope), and cortisol awakening response. Mothers of children with ALL experienced emotional symptoms many months after the initial diagnosis.
doi:10.1177/1043454213520346
PMCID: PMC4353492  PMID: 24608702
acute lymphoblastic leukemia; ALL; depression; maintenance; salivary
11.  Feasibility of motivational interviewing delivered by a glaucoma educator to improve medication adherence 
Introduction
Adherence to glaucoma treatment is poor, potentially reducing therapeutic effects. A glaucoma educator was trained to use motivational interviewing (MI), a patient-centered counseling style, to improve adherence. This study was designed to evaluate whether MI was feasible in a busy ophthalmology practice.
Methods
Feasibility was assessed using five criteria from the National Institutes of Health Behavior Change consortium: fidelity of intervention components to MI theory; success of the training process; delivery of MI-consistent interventions by the glaucoma educator; patient receipt of the intervention based on enrollment, attrition, and satisfaction; and patient enactment of changes in motivation and adherence over the course of the intervention.
Results
A treatment manual was designed by a multidisciplinary team with expertise in health psychology, public health, and ophthalmology. The glaucoma educator received 6 hours of training including role-play exercises, self-study, and individual supervision. His MI-related knowledge and skills increased following training, and he delivered exclusively MI-consistent interventions in 66% of patient encounters. 86% (12/14) of eligible patients agreed to be randomized into glaucoma educator support or a control condition. All 8 patients assigned to the glaucoma educator completed at least 2 of 6 planned contacts, and 50% (4/8) completed all 6 contacts. Patients assigned to the glaucoma educator improved over time in both motivation and adherence.
Conclusion
The introduction of a glaucoma educator was feasible in a busy ophthalmology practice. Patients improved their adherence while participating in the glaucoma educator program, although this study was not designed to show a causal effect. The use of a glaucoma educator to improve glaucoma patients’ medication adherence may be feasible at other ophthalmology clinics, and can be implemented with a standardized training approach. Pilot data show the intervention can be implemented with fidelity, is acceptable to patients and providers, and has the potential to improve adherence.
doi:10.2147/OPTH.S12765
PMCID: PMC2952610  PMID: 20957054
adherence; counseling; glaucoma; medication; training
12.  Inhibition of human gamma-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors 
The Biochemical journal  2013;450(3):10.1042/BJ20121435.
SYNOPSIS
Gamma-glutamyl transpeptidase (GGT) is an essential enzyme for maintaining cysteine homeostasis, leukotriene synthesis, metabolism of glutathione-conjugates and catabolism of extracellular glutathione. Overexpression of GGT has been implicated in many pathologies, and clinical inhibitors of GGT are under development for use in the treatment of asthma, cancer and other diseases. Inhibitors are generally characterized using synthetic GGT substrates. This study of uncompetitive inhibitors of GGT, has revealed that the potency with which compounds inhibit GGT activity in the standard biochemical assay does not correlate with the potency with which they inhibit the physiological reaction catalyzed by GGT. Kinetic studies provided insight into the mechanism of inhibition. Modifications to the sulfobenzene or distal benzene ring of the uncompetitive inhibitor, OU749, affected activity. One of the most potent inhibitors was identified among a novel group of analogs with an amine group para on the benzosulfonamide ring. New, more potent uncompetitive inhibitors of the physiological GGT reaction were found to be less toxic than the glutamine-analogs that have been tested clinically. Development of non-toxic inhibitors is essential for exploiting GGT as a therapeutic target.
doi:10.1042/BJ20121435
PMCID: PMC3836663  PMID: 23301618
Glutathione; gamma-glutamyl transferase; OU749
13.  Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target 
PLoS ONE  2013;8(10):e77558.
The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.
doi:10.1371/journal.pone.0077558
PMCID: PMC3805590  PMID: 24167577
14.  Divergent Effects of Compounds on the Hydrolysis and Transpeptidation Reactions of Gamma-Glutamyl Transpeptidase 
A novel class of inhibitors of the enzyme gamma-glutamyl transpeptidase (GGT) were evaluated. OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.
doi:10.3109/14756366.2011.597748
PMCID: PMC3407035  PMID: 21864033
Gamma-glutamyl substrates; OU749; Enzyme; Inhibition
15.  Evidence in Support of Lysine 77 and Histidine 96 as Acid-base Catalytic Residues in Saccharopine Dehydrogenase from Saccharomyces cerevisiae 
Biochemistry  2012;51(4):857-866.
Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α–aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-Kg) using NAD+ as an oxidant. The enzyme utilizes a general acid-base mechanism to carry out its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo-form and a closed form of the enzyme with saccharopine and NADH bound have been solved at 2.0 Å and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II is observed that closes the active site cleft between the two domains and brings H96 and K77 in close proximity to the substrate binding site. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77 to M, H96 to Q single and the K77M/H96Q double mutant enzymes provide data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/Et and >103 increases in V2/KLysEt and V2/Kα-KgEt (the double mutation gives >105-fold decreases in the second order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/KLys. A value of 2.0 was also observed for D(V2/KLys)D2O when the primary deuterium kinetic isotope effect was repeated in D2O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V2/KLys indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V2/KLysEt is pH independent consistent with the loss of an acid-base catalyst and perturbation of the pKa of the second catalytic group to higher pH, likely a result of a change in the overall charge in the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H2O or D2O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH-rate profile for V2/KLysEt exhibits the pKa for K77, perturbed to a value of about 9, which must be unprotonated in order to accept a proton from the ε-amine of the substrate Lys so that it can act as a nucleophile. Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the ε-amine of the substrate lysine prior to nucleophilic attack on the α-oxo group of α-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in formation of the imine between the ε-amine of lysine and the α-oxo group of α-ketoglutarate.
doi:10.1021/bi201808u
PMCID: PMC3297426  PMID: 22243403
16.  Kinetic and Chemical Mechanisms of Homocitrate Synthase from Thermus thermophilus* 
The Journal of Biological Chemistry  2011;286(33):29428-29439.
The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg2+ or Mn2+ capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with α-ketoglutarate (α-Kg) binding prior to acetyl-CoA (AcCoA) with Mn2+, whereas it is steady state random with Mg2+, suggesting a difference in the competence of the E·Mn·α-Kg·AcCoA and E·Mg·α-Kg·AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d3) in the presence of Mg2+ is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg2+ or Mn2+ as the divalent metal ion, whereas V/Kα-Kg (with Mn2+) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as α-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.
doi:10.1074/jbc.M111.246355
PMCID: PMC3190748  PMID: 21733842
Acetyl Coenzyme A; Cooperativity; Enzyme Catalysis; Enzyme Mechanisms; Isotope Effects; Homocitrate Synthase; Metalloenzyme
17.  Gamma-Glutamyl Compounds: Substrate Specificity of Gamma-Glutamyl Transpeptidase Enzymes 
Analytical biochemistry  2011;414(2):208-214.
Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites and neuroactive compounds. Two cell surface enzymes have been identified that metabolize gamma-glutamyl compounds, gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetics analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative and is conducted at physiologic pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The Kms for reduced glutathione were 11μM for both GGT1 and GGT5. However, the Km for oxidized glutathione was 9μM for GGT1 and 43μM for GGT5. Our data show that the Kms for leukotriene C4 are equivalent for GGT1 and GGT5 at 10.8μM and 10.2μM, respectively. This assay was also used to evaluate serine-borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism and other pathways that involve gamma-glutamyl compounds.
doi:10.1016/j.ab.2011.03.026
PMCID: PMC3099546  PMID: 21447318
Glutathione; gamma-glutamyl substrates; gamma-glutamyl transpeptidase; gamma-glutamyl leukotrienase; glutamate assay
18.  Kinetics of Activated Thrombin-activatable Fibrinolysis Inhibitor (TAFIa)-catalyzed Cleavage of C-terminal Lysine Residues of Fibrin Degradation Products and Removal of Plasminogen-binding Sites* 
The Journal of Biological Chemistry  2011;286(22):19280-19286.
Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the kcat and Km of Glu1-plasminogen (Glu-Pg)-binding site removal are 2.34 s−1 and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm−1 s−1. The corresponding values of Lys77/Lys78-plasminogen (Lys-Pg)-binding site removal are 0.89 s−1 and 96 nm implying a catalytic efficiency of 9.23 μm−1 s−1. These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm−1 s−1. Interestingly, this value increases to 3.85 μm−1 s−1 in the presence of Glu-Pg. These changes are due to a decrease in Km. This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.
doi:10.1074/jbc.M110.215061
PMCID: PMC3103306  PMID: 21467042
Carboxypeptidase; Enzyme Catalysis; Fibrin; Fibrinolysis; Hemostasis; TAFIa; Carboxypeptidase U
19.  Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation☆ 
Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.
doi:10.1016/j.abb.2010.11.018
PMCID: PMC3049928  PMID: 21111704
Human; Sulfotransferase; Kinetic mechanism; Initial velocity studies
20.  Relationships among optimism, well-being, self-transcendence, coping, and social support in women during treatment for breast cancer 
Psycho-oncology  2009;18(7):716-726.
Objective
The impact of diagnosis and treatment for breast cancer, stressors that affect emotional well-being, is influenced by several psychosocial factors and the relationships among them. The purpose of this study was to investigate the relationship between optimism and emotional well-being (EWB) and the individual and combined mediation of this relationship by perceived social support (SS), problem focused coping (PFC), and self-transcendence in women with breast cancer during radiation therapy.
Methods
Ninety-three women receiving radiation treatment for breast cancer completed questionnaires that measured EWB, optimism, SS, PFC, and self-transcendence.
Results
Correlational and multiple regression analysis revealed that optimism was positively related to EWB. Of the three mediators, self-transcendence alone was found to partially mediate the relationship between optimism and EWB. The relationship between optimism and PFC was not significant. Optimism was related to SS, but its indirect effect on EWB through SS did not reach significance.
Conclusions and implications
During breast cancer treatment, the positive effects of optimism on EWB are partially mediated by a woman’s level of self-transcendence. Brief screening of women’s optimism may help identify women at risk for psychological distress. Early detection and interventions to promote psychological adjustment throughout the cancer trajectory (e.g. enhancing self-transcendence) should receive attention in future research.
doi:10.1002/pon.1461
PMCID: PMC3152259  PMID: 19034884
breast cancer; psychological distress; optimism; emotional well-being; self-transcendence
21.  Glutamates 78 and 122 in the Active Site of Saccharopine Dehydrogenase Contribute to Reactant Binding and Modulate the Basicity of the Acid-Base Catalysts* 
The Journal of Biological Chemistry  2010;285(27):20756-20768.
Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and α-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu78 and Glu122, on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pKa values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of α-ketoglutarate to E and E·NADH. Large changes in V/KLys, but not V, suggest that Glu78 and Glu122 contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pKa values observed in the V/KLys pH-rate profile of the mutant enzymes suggests that the presence of Glu78 and Glu122 modulates the basicity of the catalytic groups.
doi:10.1074/jbc.M110.119826
PMCID: PMC2898324  PMID: 20427272
Enzyme Catalysis; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Mutation; Isotope Effects; Acid-Base Chemistry; Lysine Biosynthesis; Saccharopine Dehydrogenase
22.  A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex* 
The Journal of Biological Chemistry  2010;285(17):12813-12822.
The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5′-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.
doi:10.1074/jbc.M109.075762
PMCID: PMC2857139  PMID: 20164178
Enzymes/Pyridoxal Phosphate; Metabolism/Sulfur; Methods/Fluorescence; Protein/Protein-Protein Interactions; Vitamins and Cofactors/Pyridoxal Phosphate; Stopped-flow Spectroscopy
23.  Haloacetamidine-based inactivators of Protein Arginine Deiminase 4 (PAD4): Evidence that General Acid Catalysis Promotes Efficient Inactivation** 
Dysregulated Protein Arginine Deiminase (PAD) activity, particularly PAD4, has been suggested to play a role in the onset and progression of numerous human diseases, including Rheumatoid Arthritis (RA). Given the potential role of PAD4 in RA, we set out to develop inhibitors/inactivators that could be used to modulate PAD activity and disease progression. This effort led to the discovery of two mechanism-based inactivators, denoted F- and Cl-amidine, that inactivate PAD4 via the covalent modification of an active site cysteine that is critical for catalysis. To gain further insights into the mechanism of inactivation by these compounds, the effect of pH on the rates of inactivation were determined. These results, combined with the results of solvent isotope effect and proton inventory studies, strongly suggest that the inactivation of PAD4 by F- and Cl-amidine proceeds via a multi-step mechanism that involves the protonation and stabilization of the tetrahedral intermediate formed upon nucleophilic attack by the active site cysteine, i.e. Cys645. Stabilization of this intermediate would help to drive the halide-displacement reaction, which results in the formation of a three-membered sulfonium ring that ultimately collapses to form the inactivated enzyme. This finding - that protonation of the tetrahedral intermediate is important for enzyme inactivation - may also suggest that during catalysis, protonation of the analogous intermediate is required for efficient substrate turnover.
doi:10.1002/cbic.200900698
PMCID: PMC3056394  PMID: 20014086
Deiminase; Citrulline; Cl-amidine; rheumatoid arthritis; inactivator
24.  Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature 
Journal of medicinal chemistry  2010;53(1):345-356.
The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing L-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. 400 MNXXI pentapeptides were generated, docked into OASS active site using GOLD and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4, and to a lesser extent, at position P3, also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptides-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.
doi:10.1021/jm901325e
PMCID: PMC2804909  PMID: 19928859
25.  para-Nitrophenyl Sulfate Activation of Human Sulfotransferase 1A1 Is Consistent with Intercepting the E·PAP Complex and Reformation of E·PAPS* 
The Journal of Biological Chemistry  2009;284(43):29357-29364.
Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biological activities of various biosignaling molecules and metabolizes hydroxyl-containing drugs and xenobiotics. The universal sulfuryl group donor for SULT-catalyzed sulfation is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), whereas the reaction products are a sulfated product and adenosine 3′,5′-diphosphate (PAP). Although SULT-catalyzed kinetic mechanisms have been studied since the 1980s, they remain unclear. Human SULT1A1 is an important phase II drug-metabolizing enzyme. Previously, isotope exchange at equilibrium indicated steady-state ordered mechanism with PAPS and PAP binding to the free SULT1A1 (Tyapochkin, E., Cook, P. F., and Chen, G. (2008) Biochemistry 47, 11894–11899). On the basis of activation of SULT1A1 by para-nitrophenyl sulfate (pNPS), an ordered bypass mechanism has been proposed where pNPS sulfates PAP prior to its release from the E·PAP complex regenerating E·PAPS. Data are consistent with uncompetitive substrate inhibition by naphthol as a result of formation of the E·PAP·naphthol dead-end complex; formation of the complex is corroborated by naphthol/PAP double inhibition experiments. pNPS activation data demonstrate an apparent ping-pong behavior with pNPS adding to E·PAP, and competitive inhibition by naphthol consistent with formation of the E·PAP·naphthol complex. Exchange against forward reaction flux (PAPS plus naphthol) beginning with [35S]PAPS and generating [35S]naphthyl sulfate is also consistent with pNPS intercepting the E·PAP complex. Overall, data are consistent with the proposed ordered bypass mechanism.
doi:10.1074/jbc.M109.049312
PMCID: PMC2785567  PMID: 19706609

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