Kinetics of substrate and product interactions with arsanilazotyrosine-248 carboxypeptidase A

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Abstract
The chromophoric intramolecular azoTyr-248.cntdot.Zn complex detects discrete kinetic steps in the interaction of azocarboxypeptidase with products or substrates that are hydrolyzed slowly. Temperature-jump experiments at 510 nm indicate that the rapid binding of such ligands is followed by a slower change in the conformation of the enzyme-ligand complex: .**GRAPHIC**. Analysis of the data generates K1, the equilibrium constant that defines the initial binding, and the rate constants k2 and k-2 for the forward and reverse steps of this conversion, respectively. For each ligand, the kinetically determined dissociation constant is virtually identical to that obtained at equilibrium from circular dichroic titrations. Although there are small variations in k2 and k-2 for each substrate, all the rate processes are much faster than the rate-determining step for the hydrolysis of these substrates. The proposed model of the mechanism of peptide hydrolysis of carboxypeptidase incorporates the results of these temperature jump experiments.

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