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

详细信息    查看全文

• 作者：James Solowiej ; James A. Thomson ; Kevin Ryan ; Chun Luo ; Mingying He ; Jihong Lou ; Brion W. Murray
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：February 26, 2008
• 年：2008
• 卷：47
• 期：8
• 页码：2617 - 2630
• 全文大小：185K
• 年卷期：v.47,no.8(February 26, 2008)
• ISSN：1520-4995

文摘

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirusthat has a cysteine protease (3CL^pro) essential to its life cycle. Steady-state and pre-steady-state kineticmethods were used with highly active 3CL^pro to characterize the reaction mechanism. We show that 3CL^prohas mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin.The kinetic mechanism for 3CL^pro-mediated ester hydrolysis, including the individual rate constants, isconsistent with a simple double displacement mechanism. The pre-steady-state burst rate was independentof ester substrate concentration indicating a high commitment to catalysis. When homologous peptidicamide 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 havesimilar 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 thepre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studieson amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition statewhile the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivationprofile of 3CL^pro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data areconsistent with a 3CL^pro 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.