The effect of monovalent cations on the pre-steady state reaction kinetics of bovine activated plasma protein C and des-1-41-light chain activated plasma protein C.

K A Hill,F J Castellino

doi:10.1016/s0021-9258(19)75900-6

Abstract

A pre-steady state kinetic analysis of the stimulation by monovalent cations of the activity of bovine activated protein C (APC) and a proteolytic fragment of APC, des-1-41-light chain activated protein C (GDAPC), toward the substrate, 4-methylumbelliferyl p-guanidinobenzoate, has been undertaken. With the cations Na+ and Cs+, at least two cation sites, or classes of sites, on APC were found to be important to the kinetic effects observed. For GDAPC, with both monovalent cations investigated, a single cation-binding site, or class of sites, of kinetic importance was discovered. The most general mechanism that fits all kinetic data was a rapid equilibrium type, with the cation(s) (A) and substrate (S) binding to the enzyme in a random fashion. Cations were found to be essential activators, and only formation of the EAS or EA2S complex led to product generation. For each enzyme, stimulation of the reaction rates was found to be chiefly due to a dramatic enhancement by monovalent cations of the rate constant (k2) for acylation of the enzyme since the dissociation constant (Ks) for enzyme-substrate interactions was increased in the presence of cations, and the deacylation rate constant (k3) was not affected by these activators.

Highlights

A pre-steadystate kinetic analysisof the stimulation linked polypeptide chains
Upon addition of GDAPC, the trace shows that an initial burst of substrate hydrolysis occurs, corresponding to the transient phase of the reaction, followed by a portion where hydrolysis becomes linear with time
Doublereciprocal plots of the pseudo first-order rate constants of MUGB hydrolysis for the Na+- and(2s'-induced stimulation of APC have beepnlotted against[MUGB].The dataobtained at varying cation concentrations were qualitatively similar to those for GDAPC, except for Na+

Summary

EXPERIMENTAL PROCEDURES

Proteins-Bovine PC was isolated from fresh bovine plasma by modification (14) of the procedure of Stenflo (18),with the final purification by preparative polyacrylamide gel electrophoresis similar tothat described by Kisiel and Davie (19). Upon addition of GDAPC, the trace shows that an initial burst of substrate hydrolysis occurs, corresponding to the transient phase of the reaction, followed by a portion where hydrolysis becomes linear with time This linear portion has been extended in the upper dashed line and represents the sum of steady state enzymatic turnoverand spontaneous hydrolysis of MUGB. For analysis of the data according to the above scheme, we have assumed that the ES complexdoes form, but does not lead to significant amountsof product formation ( k , approaches zero) and that only the EAS complex can generate sient phase curve from steady state kinetics This was measured experimentally as thechange in fluorescence,calibrated to A[HMC], between the trace generated by the chartrecorder and the hypothetical steady state line. The illustrations presented are from single experiments, at least three such determinations were employed to generatevalues for the rate constants

RESULTS

Findings

DISCUSSION