Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL super(pro) Cysteine Protease: Development of an Ion-Pair Model for Catalysis

• Published in: Biochemistry (Easton) Vol. 47; no. 8; pp. 2617 - 2630
• Main Authors: Solowiej, James, Thomson, James A, Ryan, Kevin, Luo, Chun, He, Mingying, Lou, Jihong, Murray, Brion W
• Format: Journal Article
• Language: English
• Published: 01.01.2008
• Subjects: SARS coronavirus
• ISSN: 0006-2960
• DOI: 10.1021/bi702107vPII:S0006-2960(70)02107-4

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CL super(pro)) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CL super(pro) to characterize the reaction mechanism. We show that 3CL super(pro) has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CL super(pro)- 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 3CL super(pro) with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CL super(pro) mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P sub(1), and a general base-catalyzed deacylation reaction.