Chemical Mechanism of a Cysteine Protease, Cathepsin C, As Revealed by Integration of both Steady-State and Pre-Steady-State Solvent Kinetic Isotope Effects

Biochemistry ◽  
2008 ◽  
Vol 47 (33) ◽  
pp. 8697-8710 ◽  
Author(s):  
Jessica L. Schneck ◽  
James P. Villa ◽  
Patrick McDevitt ◽  
Michael S. McQueney ◽  
Sara H. Thrall ◽  
...  
Keyword(s):  
Steady State ◽  
Cysteine Protease ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Chemical Mechanism ◽  
Cathepsin C ◽  
Kinetic Isotope

scholarly journals Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36

2016 ◽  
Vol 113 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
Myles B. Poulin ◽  
Jessica L. Schneck ◽  
Rosalie E. Matico ◽  
Patrick J. McDevitt ◽  
Michael J. Huddleston ◽  
...  
Keyword(s):  
Transition State ◽  
Isotope Effects ◽  
Histone H3 ◽  
Kinetic Isotope Effects ◽  
Chemical Mechanism ◽  
State Structure ◽  
Transition State Structure ◽  
Chemical Modeling ◽  
Kinetic Isotope ◽  
Human Multiple Myeloma

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf–Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-14C and 36S and secondary Me-3H3, Me-2H3, 5′-14C, and 5′-3H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

scholarly journals Kinetic isotope effects and ligand binding in PQQ-dependent methanol dehydrogenase

2005 ◽  
Vol 388 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Parvinder HOTHI ◽  
Michael J. SUTCLIFFE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Steady State ◽  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Competitive Binding ◽  
Catalytic Site ◽  
Methanol Dehydrogenase ◽  
Ammonium Concentration ◽  
Steric Interaction ◽  
Kinetic Isotope

The reaction of PQQ (2,7,9-tricarboxypyrroloquinoline quinone)-dependent MDH (methanol dehydrogenase) from Methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [GEE (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of MDH). Studies of initial velocity with methanol (protiated methanol, C1H3O1H) and [2H]methanol (deuteriated methanol, C2H3O2H) as substrate, performed with different concentrations of GEE and PES (phenazine ethosulphate), indicate competitive binding effects for substrate and PES on the stimulation and inhibition of enzyme activity by GEE. GEE is more effective at stimulating activity than ammonium at low concentrations, suggesting tighter binding of GEE to the active site. Inhibition of activity at high GEE concentration is less pronounced than at high ammonium concentration. This suggests a close spatial relationship between the stimulatory (KS) and inhibitory (KI) binding sites in that binding of GEE to the KS site sterically impairs the binding of GEE to the KI site. The binding of GEE is also competitive with the binding of PES, and GEE is more effective than ammonium in competing with PES. This competitive binding of GEE and PES lowers the effective concentration of PES at the site competent for electron transfer. Accordingly, the oxidative half-reaction, which is second-order with respect to PES concentration, is more rate-limiting in steady-state turnover with GEE than with ammonium. The smaller methanol C-1H/C-2H kinetic isotope effects observed with GEE are consistent with a larger contribution made by the oxidative half-reaction to rate limitation. Cyanide is much less effective at suppressing ‘endogenous’ activity in the presence of GEE than with ammonium, which is attributed to impaired binding of cyanide to the catalytic site through steric interaction with GEE bound at the KS site. The kinetic model developed previously for reactions of MDH with ammonium [Hothi, Basran, Sutcliffe and Scrutton (2003) Biochemistry 42, 3966–3978] is consistent with data obtained with GEE, although a more detailed structural interpretation is given here. Molecular-modelling studies rationalize the kinetic observations in terms of a complex binding scenario at the molecular level involving two spatially distinct inhibitory sites (KI and KI′). The KI′ site caps the entrance to the active site and is interpreted as the PES binding site. The KI site is adjacent to, and, for GEE, overlaps with, the KS site, and is located in the active-site cavity close to the PQQ cofactor and the catalytic site for methanol oxidation.

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