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1.  Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs 
Journal of molecular biology  2008;376(5):1438-1450.
Summary
Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate (NAAG) and folyl-poly-γ-glutamates, respectively. During the past years, tremendous efforts have been made towards the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1′ site of the enzyme. In this paper, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1′ sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-γ-glutamate, aspartyl-glutamate and γ-glutamyl-glutamate, reined at resolution of 1.50 Å, 1.60 Å and 1.67 Å, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity towards different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an excellent foundation for the structurally aided design of novel low-molecular weight GCPII inhibitors and imaging agents.
doi:10.1016/j.jmb.2007.12.066
PMCID: PMC2753231  PMID: 18234225
prostate-specific membrane antigen; metallopeptidase; folate hydrolase; NAALADase; phosphapeptide
2.  Glutamate Carboxypeptidase II in Diagnosis and Treatment of Neurologic Disorders and Prostate Cancer 
Current Medicinal Chemistry  2012;19(6):856-870.
Glutamate carboxypeptidase II (GCPII) is a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. Throughout the nervous system, glia-bound GCPII is intimately involved in the neuron-neuron and neuron-glia signaling via the hydrolysis of N-acetylaspartylglutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. The inhibition of the GCPII-controlled NAAG catabolism has been shown to attenuate neurotoxicity associated with enhanced glutamate transmission and GCPII-specific inhibitors demonstrate efficacy in multiple preclinical models including traumatic brain injury, stroke, neuropathic and inflammatory pain, amyotrophic lateral sclerosis, and schizophrenia. The second major area of pharmacological interventions targeting GCPII focuses on prostate carcinoma; GCPII expression levels are highly increased in androgen-independent and metastatic disease. Consequently, the enzyme serves as a potential target for imaging and therapy. This review offers a summary of GCPII structure, physiological functions in healthy tissues, and its association with various pathologies. The review also outlines the development of GCPII-specific small-molecule compounds and their use in preclinical and clinical settings.
PMCID: PMC3341092  PMID: 22214450
Metalloprotease; prostate-specific membrane antigen; glutamate excitotoxicity; prostate cancer; N-acetylaspartylglutamate
3.  Glutamate carboxypeptidase II and folate deficiencies result in reciprocal protection against cognitive and social deficits in mice: implications for neurodevelopmental disorders 
Developmental Neurobiology  2012;72(6):891-905.
Interactions between genetic and environmental risk factors underlie a number of neuropsychiatric disorders, including schizophrenia (SZ) and autism (AD). Due to the complexity and multitude of the genetic and environmental factors attributed to these disorders, recent research strategies focus on elucidating the common molecular pathways through which these multiple risk factors may function. In this study, we examine the combined effects of a haplo-insufficiency of glutamate carboxypeptidase II (GCPII) and dietary folic acid deficiency. In addition to serving as a neuropeptidase, GCPII catalyzes the absorption of folate. GCPII and folate depletion interact within the one-carbon metabolic pathway and/or of modulate the glutamatergic system. Four groups of mice were tested: wildtype, GCPII hypomorphs, and wildtypes and GCPII hypomorphs both fed a folate deficient diet. Due to sex differences in the prevalence of SZ and AD, both male and female mice were assessed on a number of behavioral tasks including locomotor activity, rotorod, social interaction, pre-pulse inhibition, and spatial memory. Wildtype mice of both sexes fed a folic acid deficient diet showed motor coordination impairments and cognitive deficits, while social interactions were decreased only in males. GCPII mutant mice of both sexes also exhibited reduced social propensities. In contrast, all folate-depleted GCPII hypomorphs performed similarly to untreated wildtype mice, suggesting that reduced GCPII expression and folate deficiency are mutually protective. Analyses of folate and neurometabolite levels associated with glutamatergic function suggest several potential mechanisms through which GCPII and folate may be interacting to create this protective effect.
doi:10.1002/dneu.21000
PMCID: PMC3401954  PMID: 22076974
gene-environment interactions; glutamate; glutamate carboxypeptidase II; folate; one-carbon metabolism
4.  A high-resolution structure of ligand-free human glutamate carboxypeptidase II 
A crystal structure of ligand-free human glutamate carboxypeptidase II refined to 1.65 Å resolution is reported. The structure provides insight into the active-site of the enzyme in its unliganded state.
Human glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) is an established marker for prostate-cancer diagnosis as well as a candidate therapeutic target for the treatment of diverse pathologies that involve glutamatergic transmission. Structural data on GCPII are thus valuable for the design and optimization of GCPII-specific inhibitors and diagnostic probes. The currently available structure of ligand-free GCPII was refined to a resolution of 3.5 Å. This work reports the structure of the protein refined to 1.65 Å resolution, with crystallographic values of R = 0.207 and R free = 0.228. The new structure extends the resolution appreciably and the new model based on this data shows significant differences when compared with the previously published model.
doi:10.1107/S174430910700379X
PMCID: PMC2330195  PMID: 17329803
prostate-specific membrane antigens; metallopeptidase; folate hydrolases; NAALADase
5.  Bioisosterism of Urea-Based GCPII Inhibitors: Synthesis and Structure-Activity Relationships Studies 
We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure-activity relationship studies of the P1’ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with Ki values below 20 nM. Among them, compound 32d (Ki = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1’ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d.
doi:10.1016/j.bmcl.2009.10.061
PMCID: PMC2818328  PMID: 19897367
PSMA; glutamate carboxypeptidase II; molecular imaging; radiopharmaceutical; SPECT
6.  Glutamate carboxypeptidase II (GCPII) inhibitor displays anti-glutamate and anti-cocaine effects in an invertebrate assay 
Amino Acids  2011;42(6):2521-2524.
Summary
Glutamate carboxypeptidase II (GCPII) inhibitors are promising anti-glutamatergic and anti-addictive agents. We hypothesized that a GCPII inhibitor 2 (phosphonomethyl)pentanedioic acid (2-PMPA) would display anti-stereotypical activity in planarians. Experiments revealed that 2-PMPA displayed no overt behavioral activity by itself but attenuated stereotypical counts (C-shape hyperkinesias) elicited by four compounds (2-PMPA rank order potency: glutamate > NMDA > pilocarpine > cocaine). These data suggest GCPII inhibitors display broad-spectrum efficacy against behavioral activity produced by glutamatergic and non-glutamatergic compounds in an invertebrate assay.
doi:10.1007/s00726-011-1052-6
PMCID: PMC3265618  PMID: 21850438
GCPII; glutamate; 2-PMPA; cocaine; planaria; NMDA; pilocarpine; stereotypy
7.  NAAG peptidase inhibitors block cognitive deficit induced by MK-801 and motor activation induced by d-amphetamine in animal models of schizophrenia 
Translational Psychiatry  2012;2(7):e145-.
The most widely validated animal models of the positive, negative and cognitive symptoms of schizophrenia involve administration of d-amphetamine or the open channel NMDA receptor blockers, dizocilpine (MK-801), phencyclidine (PCP) and ketamine. The drug ZJ43 potently inhibits glutamate carboxypeptidase II (GCPII), an enzyme that inactivates the peptide transmitter N-acetylaspartylglutamate (NAAG) and reduces positive and negative behaviors induced by PCP in several of these models. NAAG is an agonist at the metabotropic glutamate receptor 3 (mGluR3). Polymorphisms in this receptor have been associated with expression of schizophrenia. This study aimed to determine whether two different NAAG peptidase inhibitors are effective in dopamine models, whether their efficacy was eliminated in GCPII knockout mice and whether the efficacy of these inhibitors extended to MK-801-induced cognitive deficits as assessed using the novel object recognition test. ZJ43 blocked motor activation when given before or after d-amphetamine treatment. (R,S)-2-phosphono-methylpentanedioic acid (2-PMPA), another potent NAAG peptidase inhibitor, also reduced motor activation induced by PCP or d-amphetamine. 2-PMPA was not effective in GCPII knockout mice. ZJ43 and 2-PMPA also blocked MK-801-induced deficits in novel object recognition when given before, but not after, the acquisition trial. The group II mGluR antagonist LY341495 blocked the effects of NAAG peptidase inhibition in these studies. 2-PMPA was more potent than ZJ43 in a test of NAAG peptidase inhibition in vivo. By bridging the dopamine and glutamate theories of schizophrenia with two structurally different NAAG peptidase inhibitors and demonstrating their efficacy in blocking MK-801-induced memory deficits, these data advance the concept that NAAG peptidase inhibition represents a potentially novel antipsychotic therapy.
doi:10.1038/tp.2012.68
PMCID: PMC3410622  PMID: 22850437
cognition; d-amphetamine; NAAG; N-acetylaspartylglutamate; PCP; schizophrenia
8.  Dysregulation of Glutamate Carboxypeptidase II in Psychiatric Disease 
Schizophrenia research  2008;99(1-3):324-332.
Experimental evidence is beginning to converge on an important role for dysregulation of glutamate carboxypeptidase II (GCPII) in schizophrenia. The goal of this study was to determine GCPII levels in postmortem brain specimens of patients with schizophrenia, bipolar disorder or unipolar depression and age-matched control subjects. We used N-[N(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-[125I]iodo-l-tyrosine ([125I]DCIT), a high-affinity radioligand for GCPII, to probe for GCPII expression in prefrontal cortex (PFC) and mesial temporal lobe, two brain regions implicated in the pathophysiology of schizophrenia. We found that GCPII levels measured by [125I]DCIT quantitative autoradiography were significantly lower in the PFC and entorhinal cortex in patients with schizophrenia compared to age-matched controls. Patients with bipolar disorder also expressed significantly lower GCPII levels in PFC than controls. The decrease in [125I]DCIT binding in schizophrenia and bipolar disorder remained significant after adjusting for drug abuse. A significant difference in GCPII levels were also observed between schizophrenia relative to bipolar disorder and depressed subjects in the hippocampus-stratum lucidum and between schizophrenia and bipolar in the CA2 region of the hippocampus with bipolar and depressed subjects expressing higher levels of GCPII than subjects with schizophrenia. These differences in hippocampal GCPII levels may implicate differences in the etiologies of these mental disorders. In summary, this study demonstrates a regional dysregulation of GCPII expression in the brain of patients with schizophrenia and other psychiatric disorders and supports a hypoglutamatergic state of the former illness. GCPII may represent a viable therapeutic target for intervention in psychiatric disease. (241 words)
doi:10.1016/j.schres.2007.11.013
PMCID: PMC2287371  PMID: 18191545
Schizophrenia; bipolar disorder; depression; NAALADase; GCPII - [125I]DCIT, human brain; imaging
9.  NAAG Peptidase Inhibitor Reduces Cellular Damage in a Model of TBI with Secondary Hypoxia 
Brain research  2012;1469:144-152.
Traumatic brain injury (TBI) leads to a rapid and excessive glutamate elevation in the extracellular milieu, resulting in neuronal degeneration and astrocyte damage. Posttraumatic hypoxia is a clinically relevant secondary insult that increases the magnitude and duration of glutamate release following TBI. N-acetyl-aspartyl glutamate (NAAG), a prevalent neuropeptide in the CNS, suppresses presynaptic glutamate release by its action at the mGluR3 (a group II metabotropic glutamate receptor). However, extracellular NAAG is rapidly converted into NAA and glutamate by the catalytic enzyme glutamate carboxypeptidase II (GCPII) reducing presynaptic inhibition. We previously reported that the GCPII inhibitor ZJ-43 and its prodrug di-ester PGI-02776 reduce the deleterious effects of excessive extracellular glutamate when injected systemically within the first 30 minutes following injury. We now report that PGI-02776 (10 mg/kg) is neuroprotective when administered 30-minutes post-injury in a model of TBI plus 30 minutes of hypoxia (FiO2 = 11%). 24-hrs following TBI with hypoxia, significant increases in neuronal cell death in the CA1, CA2/3, CA3c, hilus and dentate gyrus were observed in the ipsilateral hippocampus. Additionally, there was a significant reduction in the number of astrocytes in the ipsilateral CA1, CA2/3 and in the CA3c/hilus/dentate gyrus. Administration of PGI-02776 immediately following the cessation of hypoxia significantly reduced neuronal and astrocytic cell death across all regions of the hippocampus. These findings indicate that NAAG peptidase inhibitors administered post-injury can significantly reduce the deleterious effects of TBI combined with a secondary hypoxic insult.
doi:10.1016/j.brainres.2012.06.021
PMCID: PMC3424068  PMID: 22750589
Traumatic brain injury (TBI); Hypoxia; Hippocampus; Neuronal degeneration; Glutamate; N-acetylaspartylglutamate (NAAG); astrocyte
10.  Localization of NAAG-related gene expression deficits to the anterior hippocampus in schizophrenia 
Schizophrenia research  2009;111(1-3):131-137.
N-acetyl aspartyl glutamate (NAAG) is an endogenous agonist at the metabotropic glutamate receptor 3 (mGluR3,GRM3) receptor and antagonist at the N-methyl D-aspartate (NMDA) receptor, both receptors important to the pathophysiology of schizophrenia. Glutamate carboxypeptidase II (GCPII), an enzyme that metabolizes NAAG, is also implicated in this illness. In this study, we conducted in situ hybridization experiments to examine expression of mGluR3 and GCPII transcripts along the rostrocaudal axis of the human postmortem hippocampus. We hypothesized that we would find changes in mGluR3 and/or GCPII in the AH but not posterior hippocampus (PH) in schizophrenia. We compared mRNA levels of these genes in the dentate gyrus (DG) and cornu ammonis (CA)1 and CA3 of AH and PH in 20 matched pairs of control and schizophrenia cases. In controls, mGluR3 is highly expressed in the DG and at lower levels in CA1 and CA3 while GCP II is expressed at similar levels in these regions. Group comparisons show a significant reduction of GCPII mRNA level in the AH in schizophrenia. Post hoc analyses reveal this difference is localized to the CA1 region. In addition, we find a significant positive correlation between GCPII and mGluR3 mRNA in the CA3 of the control AH (r=0.66,p=0.008) which is not present in schizophrenia (r=0.096,p=0.76). This may reflect a disrupted functional interaction between NAAG and mGluR3 in CA3 in schizophrenia. These data suggest that NAAG-mediated signaling is disrupted in the AH in schizophrenia and localize the defect to the CA1 and CA3 regions.
doi:10.1016/j.schres.2009.03.038
PMCID: PMC2685203  PMID: 19403271
glutamate; psychosis; post mortem; in situ; GRM3; mGluR3
11.  pH-dependent Reactivity for Glycyl-L-Tyrosine in Carboxypeptidase A Catalyzed Hydrolysis 
The journal of physical chemistry. B  2011;115(34):10360-10367.
The dipeptide glycyl-L-tyrosine (GY) can be either a substrate for carboxypeptidase A (CPA) or an inhibitor, depending on pH. In this work, we investigate the pH dependent reactivity of this dipeptide in CPA catalyzed hydrolysis using a combined quantum mechanical and molecular mechanical method. It is shown that the mono-ionic form of the dipeptide, prevalent at high pH, chelates the active-site zinc ion, rendering the enzyme inactive. This inhibitory form is consistent with an earlier X-ray structure of the CPA-GY complex. On the other hand, the prevailing di-ionic form of the dipeptide at low pH was found to undergo hydrolysis via nucleophilic mechanism, leading to an acyl-enzyme complex. The stability of this reaction intermediate is consistent with previous low-temperature solid-state NMR results. The calculated overall free energy barrier of 20.1 kcal/mol is in excellent agreement with the experimental value of 19.9 kcal/mol.
doi:10.1021/jp2046504
PMCID: PMC3162075  PMID: 21732684
QM/MM; MD; carboxypeptidase A; catalytic mechanism; hydrolysis
12.  Differential expression of metabotropic glutamate receptor 2 and 3 in schizophrenia: a mechanism for antipsychotic drug action? 
The American journal of psychiatry  2009;166(7):812-820.
Objective
Preclinical and clinical data implicate the group II metabotropic glutamate receptors (mGluR2 and mGluR3) in the pathophysiology of schizophrenia. Moreover, a recent phase II clinical trial has demonstrated the antipsychotic efficacy of a mGluR2/3 agonist. The current study was designed to distinguish the expression of mGluR2 and mGluR3 receptor protein in schizophrenia and to quantify glutamate carboxypeptidase II (GCPII) in order to explore a role for the metabotropic receptors in schizophrenia therapeutics. GCPII is an enzyme that metabolizes N-acetylaspartylglutamate (NAAG), the only known specific endogenous agonist of mGluR3 in the mammalian brain.
Method
The normal expression levels of mGluR2, mGluR3 and GCPII were determined in 10 regions of the human post mortem brain using specific antibodies. Differences in expression levels of each protein were then examined in the dorsolateral prefrontal (DLPFC), temporal (TC) and motor cortex (MC) in 15 matched cases of schizophrenia and normal controls. Chronic antipsychotic treatment in rodents was conducted to examine the potential effect of antipsychotic drugs on expression of the 3 proteins.
Results
We found a significant increase in GCPII protein and a reduction in mGluR3 protein in the DLPFC in schizophrenia with mGluR2 protein levels unchanged. Chronic antipsychotic treatment in rodents did not influence GCPII or mGluR3 levels.
Conclusions
Increased GCPII expression and low mGluR3 expression in the DLPFC suggest that NAAG-mediated signaling is impaired in this brain region in schizophrenia. Further, these data implicate the mGluR3 receptor in the antipsychotic action of mGluR2/3 agonists.
doi:10.1176/appi.ajp.2009.08091445
PMCID: PMC2860261  PMID: 19487395
prefrontal cortex; glutamate carboxypeptidase II; mGluR; psychosis; human postmortem
13.  Immunohistological and electrophysiological evidence that N-acetylaspartylglutamate (NAAG) is a co-transmitter at the vertebrate neuromuscular junction 
Immunohistochemical studies previously revealed the presence of the peptide transmitter N-acetylaspartylglutamate (NAAG) in spinal motor neurons, axons and presumptive neuromuscular junctions (NMJ). At synapses in the CNS, NAAG has been shown to activate the type 3 metabotropic glutamate receptor (mGluR3) and is inactivated by an extracellular peptidase, glutamate carboxypeptidase II (GCPII). The present study tested the hypothesis that NAAG meets the criteria for classification as a cotransmitter at the vertebrate NMJ. Confocal microscopy confirmed the presence of NAAG immunoreactivity and extended the resolution of the peptide's location in the lizard (Anolis carolinensis) NMJ. NAAG was localized to a presynaptic region immediately adjacent to postsynaptic acetylcholine receptors. NAAG was depleted by potassium-induced depolarization and by electrical stimulation of motor axons. The NAAG receptor, mGluR3, was localized to the presynaptic terminal consistent with NAAG's demonstrated role as a regulator of synaptic release at central synapses. In contrast, glutamate receptors, mGluR2 and NMDA, were closely associated with acetylcholine receptors in the postsynaptic membrane. GCPII, the NAAG inactivating enzyme, was identified exclusively in perisynaptic glial cells. This localization was confirmed by the loss of immunoreactivity when these cells were selectively eliminated. Finally, electrophysiological studies showed that exogenous NAAG inhibited evoked neurotransmitter release by activating a group II metabotropic glutamate receptor (mGluR2 or mGluR3). Collectively, these data support the conclusion that NAAG is a co-transmitter at the vertebrate NMJ.
doi:10.1111/ejn.12027
PMCID: PMC3538924  PMID: 23134476
vertebrate neuromuscular junction; N-acetylaspartylglutamate; NAAG; mGluR3; GCPII; NMDA; Anolis carolinensis
14.  Combined Impact of Polymorphism of Folate Metabolism Genes; Glutamate Carboxypeptidase, Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase on Breast Cancer Susceptibility in Kashmiri Women 
Background:
Folate and methionine play a crucial role in DNA synthesis, repair and the epigenetic profile of cell. Hence, the alterations in the folate metabolism can lead to aberrant proliferation leading to neoplasia. Most of the studies have associated polymorphisms in methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes with reduced risk of cervical and colorectal cancer. However, the association with breast cancer is still controversial. Further, the involvement of Glutamate carboxypeptidase II (GCPII) polymorphism in cancer is not known. In the present study, we analyzed if the individual and combined effects of polymorphisms in folate pathway genes viz., MTHFR 677C > T, MTHFR 1298A > C, MTRR 66A > G and GCP II 1561 C>T, have any role in altering the susceptibility to breast cancer.
Methods:
The DNA of 35 female breast cancer patients and 33 healthy individuals, in the Kashmiri population from India, were analyzed using a PCR-RFLP approach for the above mentioned polymorphisms.
Results:
Individuals carrying the MTHFR 677CT/TT and GCPII 1561 CT genotype showed a 3.5 (95% CI: 3.1–3.7, P<0.02) and 7.7 (95% CI: 6.7–9.1, P<0.001) fold decreased risk for breast cancer than the wild types (MTHFR 677CC and GCPII 1561 CC). Subjects with MTRR 66 G-allele showed a 4.5 fold decreased risk (OR: 0.22, 95% CI: 0.20, 0.24, P<0.0005) compared to the wild type (MTRR 66A). Further, subjects with combined polymorphisms in MTHFR, GCPII and MTRR loci revealed a significant reduction of breast cancer risk.
Conclusion:
This study indicates (i) a protective role of polymorphisms in MTHFR, GCPII, MTRR against breast cancer in the study subjects, and (ii) combined effect of polymorphisms is more pronounced than single genetic polymorphism, thereby emphasizing the role of gene-gene interaction in the susceptibility to breast cancer.
PMCID: PMC3068715  PMID: 21475466
15.  Fluoroketone Inhibition of Ca2+-Independent Phospholipase A2 through Binding Pocket Association Defined by Hydrogen/Deuterium Exchange and Molecular Dynamics 
The mechanism of inhibition of group VIA Ca2+-independent phospholipase A2 (iPLA2) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA2 were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA2 should greatly improve our ability to design new inhibitors with higher potency and selectivity.
doi:10.1021/ja306490g
PMCID: PMC3561773  PMID: 23256506
16.  The Design of a Next Generation Force Field: The X-POL Potential 
An electronic structure-based polarization method, called the X-POL potential, has been described for the purpose of constructing an empirical force field for modeling polypeptides. In the X-POL potential, the internal, bonded interactions are fully represented by an electronic structure theory augmented with some empirical torsional terms. Non-bonded interactions are modeled by an iterative, combined quantum mechanical and molecular mechanical method, in which the molecular mechanical partial charges are derived from the molecular wave functions of the individual fragments. In this paper, the feasibility of such an electronic structure force field is illustrated by small model compounds. A method has been developed for separating a polypeptide chain into peptide units and its parameterization procedure in the X-POL potential is documented and tested on glycine dipeptide. We envision that the next generation of force fields for biomolecular polymer simulations will be developed based on electronic structure theory, which can adequately define and treat many-body polarization and charge delocalization effects.
doi:10.1021/ct700167b
PMCID: PMC2577589  PMID: 18985172
17.  An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles 
Biochemical Journal  2008;415(Pt 3):401-407.
Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated π-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity.
doi:10.1042/BJ20080440
PMCID: PMC2570083  PMID: 18412544
aliphatic nitrile; aromatic nitrile; nitrilase; Rhodococcus rhodochrous; substrate specificity; LB, Luria–Bertani
18.  The X-Ray Structure of Carboxypeptidase A Inhibited by a Thiirane Mechanism-Based Inhibitor 
Chemical biology & drug design  2009;75(1):29-34.
The three-dimensional X-ray crystal structure of carboxypeptidase A, a zinc-dependent hydrolase, covalently modified by a mechanism-based thiirane inactivator, 2-benzyl-3,4-epithiobutanoic acid, has been solved to 1.38 Å resolution. The interaction of the thiirane moiety of the inhibitor with the active site zinc ion promotes its covalent modification of Glu-270 with the attendant opening of the thiirane ring. The crystal structure determination at high resolution allowed for the clear visualization of the covalent ester bond to the glutamate side chain. The newly generated thiol from the inhibitor binds to the catalytic zinc ion in a monodentate manner, inducing a change in the zinc ion geometry and coordination, while its benzyl group fits into the S1′ specificity pocket of the enzyme. The inhibitor molecule is distorted at the position of the carbon atom that is involved in the ester bond linkage on one side and the zinc coordination on the other. This particular type of thiirane-based metalloprotease inhibitor is for the first time analyzed in complex to the target protease at high resolution and may be used as a general model for zinc-dependent proteases.
doi:10.1111/j.1747-0285.2009.00907.x
PMCID: PMC2908478  PMID: 19895506
M14 family of proteases; mechanism-based inactivation; metallopeptidase; thiirane; X-ray crystallography
19.  The action of leucyl-leucine methyl ester on cytotoxic lymphocytes requires uptake by a novel dipeptide-specific facilitated transport system and dipeptidyl peptidase I-mediated conversion to membranolytic products 
The mechanism of toxicity for cytolytic lymphocytes of Leu-Leu-OMe and related dipeptide derivatives was examined. Selective inhibition of dipeptidyl peptidase I (DPPI), a lysosomal thiol protease highly enriched in cytotoxic lymphocytes, prevented all natural killer (NK) toxic effects of such agents. However, many DPPI substrates were found to possess no NK toxic properties. For some such agents, this lack of NK toxicity appeared to be related to the lack of uptake by lymphocytes. In this regard, Leu-Leu-OMe was found to be incorporated by lymphocytes and monocytes via a saturable facilitated transport mechanism with characteristics distinct from previously characterized mammalian dipeptide transport processes. This novel transport process was found to be specific for dipeptides composed of selective L- stereoisomer amino acids and enhanced by hydrophobic ester or amide additions to the COOH terminus of dipeptides. Maximal rates of Leu-Leu- OMe uptake by T8 and NK cell-enriched peripheral blood lymphocytes (PBL) were four- to sixfold higher than for T4-enriched PBL or PBL depleted of Leu-Leu-OMe-sensitive cytotoxic lymphocytes. All dipeptide amides or esters with NK toxic properties were found to act as competitive inhibitors of [3H]Leu-Leu-OMe uptake by PBL. However, some NK nontoxic DPPI substrates were found to be comparable with Leu-Leu- OMe in avidity for this transport process. Such agents were noted to possess one or more hydrophilic amino acid side chains and were found not to mediate red blood cell lysis when subjected to the acyl transferase activity of DPPI. Thus, uptake by a dipeptide-specific facilitated transport mechanism and conversion by DPPI to hydrophobic polymerization products with membranolytic properties were found to be common features of NK toxic dipeptide derivatives. The presence of a previously unreported dipeptide transport mechanism within blood leukocytes and the selective enrichment of the granule enzyme, DPPI, within cytotoxic effector cells of lymphoid or myeloid lineage appear to afford a unique mechanism for the targeting of immunotherapeutic reagents composed of simple dipeptide esters or amides.
PMCID: PMC2188150  PMID: 1972727
20.  Mapping protein electron transfer pathways with QM/MM methods 
Journal of the Royal Society Interface  2008;5(Suppl 3):S233-S239.
Mixed quantum mechanics/molecular mechanics (QM/MM) methods offer a valuable computational tool for understanding the electron transfer pathway in protein–substrate interactions and protein–protein complexes. These hybrid methods are capable of solving the Schrödinger equation on a small subset of the protein, the quantum region, describing its electronic structure under the polarization effects of the remainder of the protein. By selectively turning on and off different residues in the quantum region, we are able to obtain the electron pathway for short- and large-range interactions. Here, we summarize recent studies involving the protein–substrate interaction in cytochrome P450 camphor, ascorbate peroxidase and cytochrome c peroxidase, and propose a novel approach for the long-range protein–protein electron transfer. The results on ascorbate peroxidase and cytochrome c peroxidase reveal the importance of the propionate groups in the electron transfer pathway. The long-range protein–protein electron transfer has been studied on the cytochrome c peroxidase–cytochrome c complex. The results indicate the importance of Phe82 and Cys81 on cytochrome c, and of Asn196, Ala194, Ala176 and His175 on cytochrome c peroxidase.
doi:10.1098/rsif.2008.0061.focus
PMCID: PMC2706105  PMID: 18445553
quantum mechanics/molecular mechanics; compound I; haem; propionates; electron transfer
21.  Gene–gene interaction in folate-related genes and risk of neural tube defects in a UK population 
Journal of Medical Genetics  2004;41(4):256-260.
Objective: To investigate the contribution of polymorphic variation in genes involved in the folate-dependent homocysteine pathway in the aetiology of neural tube defects (NTD).
Design: Case-control association study.
Subjects: A total of 530 individuals from families affected by NTD, 645 maternal controls, and 602 healthy newborn controls from the northern UK.
Main outcome measures: Seven polymorphisms in six genes coding for proteins in the folate-dependent homocysteine pathway (MTHFR 677C→T, MTHFR 1298A→C, MTRR 66A→G, SHMT 1420C→T, CßS 844ins68, GCPII 1561C→T, RFC-1 80G→A). The impact of each polymorphism and the effect of gene–gene interactions (epistasis) upon risk of NTD were assessed using logistic regression analysis.
Results: The MTHFR 677C→T polymorphism was shown to represent a risk factor in NTD cases (CC v CT+TT odds ratio (OR) 2.03 [95% confidence interval (CI) 1.09, 3.79] p = 0.025) and the MTRR 66A→G polymorphism was shown to exert a protective effect in NTD cases (AA v AG+GG OR 0.31 [95% CI 0.10, 0.94] p = 0.04). When statistical tests for interaction were conducted, three genotype combinations in cases (MTRR/GCPII; MTHFR 677/CßS; MTHFR 677/MTRR) and one combination in case mothers (CßS/RFC-1) were shown to elevate NTD risk. Maternal–fetal interaction was also detected when offspring carried the MTHFR 677C→T variant and mothers carried the MTRR 66A→G variant, resulting in a significantly elevated risk of NTD.
Conclusion: Both independent genetic effects and gene–gene interaction were observed in relation to NTD risk. Multi-locus rather than single locus analysis might be preferable to gain an accurate assessment of genetic susceptibility to NTD.
doi:10.1136/jmg.2003.010694
PMCID: PMC1735724  PMID: 15060097
22.  Theoretical Study of the Catalysis of Cyanohydrin Formation by the Cyclic Dipeptide Catalyst cyclo[(S)-His-(S)-Phe] 
The Journal of organic chemistry  2009;74(4):1464-1472.
Dipeptide cyclo[(S)-His-(S)-Phe] 1, first applied by Inoue et al. in 1981, catalyzes the hydrocyanation of aromatic aldehydes very efficiently. Enantioselective autoinduction has also been reported for the process. We have employed QM (Density Functional Theory and MP2), Molecular Mechanics (MM) and Molecular Dynamics (MD) methods to (i) derive a mechanistic picture for catalysis and (ii) reveal the origin of stereochemistry and autoinduction. A dimer is proposed to be the catalytic species, in which one imidazole group is essential for the delivery of the nucleophile and the second imidazole group acts as an acid, accompanied with π-interaction for most favorable substrate binding. H-bonding via hydroxy groups is crucial for catalysis also. MD studies indicate stability of the dimer only in non-polar media, which is consistent with the need of the experimental (heterogeneous) reaction conditions to achieve high enantioselectivities. DFT and MP2 results suggest the incorporation of the product cyanohydrin via extended edge-to-face π-interaction over three aromatic units. Transition states derived from this model are in good agreement with experimental findings and enantioselectivities.
doi:10.1021/jo801958r
PMCID: PMC2662997  PMID: 19161315
23.  Cloning and characterization of the novel gene for mast cell carboxypeptidase A. 
Journal of Clinical Investigation  1992;89(1):273-282.
No gene for a hematopoietic cell carboxypeptidase has previously been characterized. Mast cell carboxypeptidase A (MC-CPA) is a prominent secretory granule marker of mast cell differentiation and phenotype. The 32-kb human MC-CPA gene was isolated, localized to chromosome 3, and found to contain 11 exons. No significant homology was found between the 5' flanking region of the MC-CPA gene and those of three rat pancreatic carboxypeptidase genes (carboxypeptidase A1 and A2, and carboxypeptidase B [CPB]). In contrast, the intron/exon organization of the MC-CPA gene was conserved, most closely resembling the CPB gene. MC-CPA is unique among carboxypeptidases in having a CPA-like substrate-binding pocket and enzymatic activity despite overall protein and gene structures more similar to CPB. Evolutionary tree analysis of the carboxypeptidase gene family showed that, before the mammalian species radiation, a common MC-CPA/CPB ancestor diverged by gene duplication from the lineage leading to CPA, and then underwent another gene duplication to form separate but similar gene structures for MC-CPA and CPB. MC-CPA mRNA was prominent in dispersed lung cells enriched for mast cells but was undetectable in other nontransformed populations of several lineages, demonstrating that transcription of MC-CPA, a novel carboxypeptidase gene, provides a specific molecular marker for mast cells among normal hematopoietic cell populations.
Images
PMCID: PMC442845  PMID: 1729276
24.  Examination of the Mechanism of Human Brain Aspartoacylase through the Binding of an Intermediate Analogue†‡ 
Biochemistry  2008;47(11):3484-3492.
Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-l-aspartate to produce l-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-l-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl-l-aspartate.
doi:10.1021/bi702400x
PMCID: PMC2666850  PMID: 18293939
25.  The Membrane-Associated Lipoprotein-9 GmpC from Staphylococcus aureus Binds the Dipeptide GlyMet via Side Chain Interactions†,‡ 
Biochemistry  2004;43(51):16193-16202.
Bacterial dipeptide ABC transporters function to import a wide range of dipeptide substrates. This ability to transport a wide variety of dipeptides is conferred by the cognate substrate binding protein (SBP) of these transporters. SBPs bind dipeptides with little regard for their amino acid content. Here, we report the 1.7 Å resolution structure of lipoprotein-9 (SA0422) of Staphylococcus aureus in complex with the dipeptide glycylmethionine. Experimental characterization of the subcellular location of the protein confirmed that SA0422 is an acylated, peripheral membrane protein. This is the first structure determined for an SBP of a Gram-positive dipeptide ABC transporter. Usually, binding of dipeptides occurs in a binding pocket that is largely hydrated and able to accommodate the side chains of several different amino acid residues. Unlike any other known SBP, lipoprotein-9 binds the side chains of the glycylmethionine dipeptide through very specific interactions. Lipoprotein-9 shares significant structural and sequence homology with the MetQ family of methionine SBP. Sequence comparisons between MetQ-like proteins and lipoprotein-9 suggest that the residues forming the tight interactions with the methionine side chains of the ligand are highly conserved between lipoprotein-9 and MetQ homologues, while the residues involved in coordinating the glycine residue are not. Modeling of the Vibrio cholerae MetQ and lipoprotein-9 binding pockets can account for lipoprotein-9 substrate specificity toward glycylmethionine. For this reason, we have designated lipoprotein-9 GmpC, for glycylmethionine binding protein.
doi:10.1021/bi048877o
PMCID: PMC2792005  PMID: 15610013

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