title: Steady-state and pre-steady-state kinetic evaluation of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro cysteine protease: development of an ion-pair model for catalysis.
abstract: Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.
authors.affiliation: Pfizer Global Research and Development, La Jolla, Pfizer Inc., 10777 Science Center Drive, San Diego, California 92121, USA.
authors.forename: James
authors.initials: J
authors.lastname: Solowiej
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
authors.forename: Kevin
authors.initials: K
authors.lastname: Ryan
authors.forename: Chun
authors.initials: C
authors.lastname: Luo
authors.forename: Mingying
authors.initials: M
authors.lastname: He
authors.forename: Jihong
authors.initials: J
authors.lastname: Lou
authors.forename: Brion W
authors.initials: BW
authors.lastname: Murray
mesh_terms: D000179:Acrylates / Q000493:pharmacokinetics; D000478:Alkylation; D002384:Catalysis; D000086782:Coronavirus 3C Proteases; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004151:Dipeptides / Q000493:pharmacokinetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics; D004795:Enzyme Stability / Q000187:drug effects; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D012997:Solvents / Q000494:pharmacology; D055672:Static Electricity; D014166:Transferases / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C528509:4-(2-((4-methoxy-1H-indole-2-carbonyl)amino)-4-methylpentanoylamino)-5-(2-oxopyrrolidin-3-yl)pent-2-enoic acid ethyl ester; D000179:Acrylates; D004151:Dipeptides; D004791:Enzyme Inhibitors; D007477:Ions; D012997:Solvents; D014764:Viral Proteins; D006639:Histidine; D014166:Transferases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases; D003545:Cysteine; D007460:Iodoacetamide