Kinetic Studies: Tyr Mutants
The changes in enzyme kinetic parameters of TOP towards four structurally distinct substrates upon removal of the hydroxyl groups of Tyr605 and Tyr612 are shown in . The Y612F mutation resulted in a marked decrease in activity, as measured by changes in kcat/Km, with respect to wild type activity towards MCA, mcaNt, and mcaGnRH1-9. The decrease was 1000-2000-fold with respect to mcaNt and MCA and 200-fold with respect to mcaGnRH1-9 and these changes were mostly due to changes in kcat. The Y605F mutation () resulted in a lesser, but still considerable, 100-fold decrease in activity towards MCA and mcaNt, and a 12-fold decrease towards GnRH1-9, again due to changes in kcat. Interestingly, the Y605F mutant did not show significant changes in kcat/Km with the mcaBk substrate; the parameters were very similar to that of the wild type. There were significant changes, however, in the kcat/Km with the double Y605/612F mutation and less change with the single Y612F, most notably due to changes in Km.
Wild type TOP has a clear preference for the mcaBk substrate over MCA and mcaNt, based on kcat/Km values ( and ). The majority of single substitutions of Ala for Gly in the loop region further increased this selectivity by considerably decreasing the activity towards the MCA and mcaNt substrates while generally having no effect or a slight improvement of activity towards mcaBk. This effect was observed for the MCA and mcaNt substrates with the G599A, G604A, and G611A mutant forms. For instance, each enzyme had decreased overall activity towards mcaNt due to decreased kcat values when compared to wild type, except for G611A which had a kcat value similar to that of wild type. In fact, both G599A and G604A had changes in Km that were consistent with the changes in kcat; about 3-fold for kcat and about 2-fold for Km. That is, changes in activity toward the mcaNt substrate were due to changes seen in both constants, although somewhat more for kcat, whereas those towards MCA were purely due to changes in kcat.
Comparison of kcat/Km of mutants to kcat/Km of wild type for three substrates. The kcat/Km for each mutant with MCA (dark gray), mcaBk (medium gray), and mcaNt (light gray) where wild type=0 on the log scale.
However, the substitution of Ala for Gly at position 603 in either single or double mutations notably altered the preference of the enzyme ( and ). G603A had the effect of creating a greater preference for the 5-residue MCA substrate and to a lesser extent for the 10-residue mcaNt substrate compared to wild type and all other single mutants. The double mutant that combines the G603A substitution with a second Ala substitution (G604A) retained increased activity towards MCA. Activity for the double mutant towards the Nt derivative did not increase compared to wild type, although its activity was notably higher than the single G604A mutant.
Although the substitution of alanine for glycine at position 603 led to enhanced activity towards MCA and mcaNt, substitution of proline for the glycine caused a significant decrease in the kcat/Km with MCA and mcaNt. The decrease in activity was ~1000 fold with MCA and ~200 fold with mcaNt, both primarily due to decreases in kcat.
Data for the loop mutants further demonstrated that the mcaBk substrate is distinct (). This substrate showed only little to no change in activity with the loop glycine mutants. Only G611A, the mutation closest to Tyr612, resulted in any substantial effect on activity towards mcaBk. The G603A and G604A mutations, both of which lie close to Y605, caused no significant change in activity towards mcaBk. It is notable that Y612F and Y605F caused modest and no change, respectively, towards this same substrate. Substitution of proline for glycine at position 603 led to significant decreases in activity for the mcaBk substrate. In contrast to the other mutants, the change for the proline substitution was entirely due to changes in Km, not in kcat.
Denaturing activity trends
We previously reported the changes in activity of two substrates (MCA and mcaBk) at low urea concentrations [20
]. Here we have expanded on that data with two additional structurally distinct and physiologically relevant, neuropeptide-based substrates (). Similar to the Tyr mutations, urea had distinct effects on mcaBk not apparent for the other substrates tested. At low urea concentrations TOP lost activity towards MCA, mcaNt, and mcaGnRH1-9
. However, the enzyme was fully active towards mcaBk even between 1 and 2 M urea. Interestingly, the trends in activity in urea paralleled the trends observed with the Y612F mutant. For mcaBk, which suffered an increase in Km
with the Y612F mutant, low urea caused an increase in Km
. Between 1-2 M urea, the
Y612F enzyme also retained marked activity towards mcaGnRH1-9
. Both MCA and mcaNt, the most sensitive to the Y612F mutation, showed the largest decrease in activity between 1-2 M urea. Above 3 M urea the enzyme lost activity to all substrates, due to enzyme denaturation and Zn(II) loss from the active site [20
Percent activity of wt TOP with substrates MCA, mcaBk, mcaGnRH1-9, and mcaNt in the presence of increasing urea (M).
To determine if the change in activity towards the MCA and mcaNt substrates were due to a change in substrate recognition by the modified enzymes, resulting in an altered cleavage site, wild type TOP and MCA were incubated for 30 minutes and the products evaluated by HPLC. Two products at absorbance 330nm were detected, suggesting a single cleavage site in the MCA substrate. Extended incubation and examination of the products of mcaNt after 90 minutes revealed four products, leading to suggestions of additional cleavage sites for the mcaNt substrate. Identical results were obtained concerning the position of cleavage sites for the Gly mutants (data not shown).
Modeling and molecular simulations of wild type and mutant TOP
By analogy to the Dcp enzyme [19
], the transition between the open (substrate free) and closed (substrate bound) forms of TOP likely occurs through a reorientation of domains I and II. Thus, a model of the closed form of TOP was created by separately fitting domains I and II of the open TOP crystal structure onto the structure of Dcp in its closed form [19
]. The TOP domains superimposed very well on the Dcp structure, with RMS deviations of 1.50 Å and 1.21 Å for domains I and II respectively. After fitting and minimization, the closed model of TOP was quite similar to that of Dcp, indicating that the two domains of TOP form relatively rigid structures that change their relative orientation by pivoting on residues 156, 351, 544, and 616 connecting the two lobes. Modeling TOP onto Dcp moved several domain II Tyr residues of TOP known to be involved in catalysis or substrate-binding [17
] into positions analogous to those of the closed DcP and thus to the appropriate distances from the active site to perform such roles (). Tyr605 and Tyr609 fall within the loop structure while Tyr612 is just at the end of the loop. The original substrate-free structure of TOP showed that Tyr612, an important catalytic residue based on mutagenesis studies [17
], is more than 8 Å from the active site. The closed form orients the phenol oxygen of this residue within hydrogen bonding distance from the carboxyl group of the scissile peptide bond in a modeled substrate. Furthermore, Tyr605 and Tyr609, both implicated in substrate-binding are shown in to be within hydrogen bonding distance from the substrate.
Molecular model of TOP used as the initial structure for MD simulations of wt, G603A, and G604A TOP with the MCA substrate shown in space filling.
Since energy minimization only allows for limited conformational sampling, we also subjected our TOP model to an MD simulation in explicit solvent in order to sample additional conformations of the substrate and the enzyme. Although all residues were allowed to move freely in these simulations, the overall enzyme structure and the loop region maintained relatively low C α RMS deviation values from our intial model throughout this trajectory (<3.0 Å and <1.5 Å, respectively). The substrate also maintained its relative position in the active site during the simulation. These data do not preclude the existence of other possible conformations further away from the starting model that were not sampled during the MD simulation. However, significant structural homology between TOP and DcP around the active site residues of domain I and the loop and Tyr residues in domain II supports out initial conformation for the model. Furthermore, the experimental effect observed for Tyr 605 and 612 mutants on enzyme activity validate the close proximity of these residues to the substrate in the model.
The MD simulations on wild type TOP and all four glycine mutants (G599A, G603A, G604A, and G611A) also provide insight into how alanine mutations affect the structure and dynamics of the loop region. All of these simulations included a MCA-like substrate in the active site (). As expected, based on the flexibility of glycine, all four alanine mutations led to decreased structural flexibility in the loop region. For example, the loop region in the wild type enzyme had an increased Cα RMS fluctuation over the final ns of the trajectories in the glycine-rich region of the loop between residues 599-604 (data not shown). As well, the wild type loop showed an ability to more readily access a wider variety of conformations. This was particularly true for the section of the loop between residues 605 and 612 that contains the tyrosine residues demonstrated to be important for catalysis in this study. This region had a greater average Cα RMS deviation (2.7 Å) from the initial model over the last ns of the simulation than observed in mutant simulations (1.2-1.95 Å). This increased conformational sampling also led the wild type simulation to show reduced hydrogen bonding between loop residues and the substrate () at the end of the simulation despite having a close proximity between tyrosine hydroxyl groups (e.g. 2-3 Å) and the substrate in the initial model.
Percent hydrogen bonding distances of mutants
Beyond reducing the flexibility of the loop, different alanine substitutions led to different hydrogen bonding patterns between tyrosine residues in the loop and the substrate (). Thus, in addition to generally decreasing flexibility the alanine mutants may restrict the loop to different conformations relative to the substrate. It would be tenuous to interpret these hydrogen bonding results too strongly in terms of catalysis since the simulations have a relatively short timescale (10-15 ns) and include a substrate-like molecule that would not necessarily mimic enzyme interactions in the transition state. For example, tyrosine residues in the loop of wild type TOP clearly have the ability to interact with the substrate during catalysis, although the loop sampled conformations farther from the substrate in the wild type simulation. Nonetheless, these results imply that conformational differences caused by different alanine substitutions could lead to differences in experimentally observed kinetic data, such as the increased activity of G603A towards MCA compared to the adjacent G604A mutation. As well, Tyr609 formed hydrogen bonds with substrate in several trajectories. It would be interesting for future studies to consider the possible role of this residue in catalysis in more detail.