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

Biochemistry ◽  
2008 ◽  
Vol 47 (8) ◽  
pp. 2617-2630 ◽  
Author(s):  
James Solowiej ◽  
James A. Thomson ◽  
Kevin Ryan ◽  
Chun Luo ◽  
Mingying He ◽  
...  
Keyword(s):  
Steady State ◽  
Severe Acute Respiratory Syndrome ◽  
Ion Pair ◽  
Kinetic Evaluation ◽  
Pair Model ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals The mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides

1973 ◽  
Vol 133 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Michael L. Sinnott ◽  
Ian J. L. Souchard
Keyword(s):  
Steady State ◽  
Isotope Effects ◽  
Ion Pair ◽  
Enzyme Mechanism ◽  
Kinetic Isotope ◽  
Deuterium Substitution ◽  
Steady State Kinetic ◽  
Hydrolysis Of ◽  
State Kinetic ◽  
Step Mechanism

1. Steady-state kinetic parameters for the β-galactosidase-catalysed hydrolysis of 13 aryl β-d-galactopyranosides show no simple dependence on aglycone acidity. 2. α-Deuterium kinetic isotope effects (kH/kD) for seven of these substrates, measured under steady-state conditions with [S]»Km, vary from 1.00 for poor substrates to 1.25 for hydrolysis of the galactosyl-enzyme. 3. Methanolysis of the galactosyl-enzyme in 1.5m-methanol increases KH/kD for degalactosylation, but leaves that for hydrolysis of ‘slow’ substrates unchanged. 4. These data are incompatible with a simple two-step mechanism. A scheme consisting of a conformation change, liberation of a galactopyranosyl cation in an intimate ion-pair, non-productive but preferential collapse of the ion-pair to a covalent species and reaction of the galactosyl enzyme through the ion-paired form is proposed. 5. This scheme is used to rationalize previously puzzling data about the enzyme mechanism.

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