Effect of monovalent cations on the pre-steady-state kinetic parameters of the plasma protease activated bovine protein C

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Abstract
Activated bovine plasma protein C (APC) was not reactive with the substrate p-nitrophenyl p-guanidinobenzoate (NPGB) in the absence of cations. In the presence of increasing concentrations of Na+, the acylation rate constant, k2,app, at 7.degree. C, progressively increased from 0.32 .+-. 0.03 s-1 at 12.5 mM Na+ to 1.15 .+-. 0.10 s-1 at 62.5 mM Na+. A linear dependence of the reciprocal of K2,app with [Na+]-2was observed, indicating that at least 2-monovalent cation sites or classes of sites, are necessary for the catalytic event to occur. From this latter plot, the k2,max for APC catalysis of NPGB hydrolysis, at saturating [Na+] and [NPGB], was calculated to be 1.21 .+-. 0.10 s-1, and Km for Na+ was found to be 21 .+-. 3 mM. The dissociation constant, Ks, for NPGB to APC, at 7.degree. C, was not altered as [Na+] was increased, yielding a range of values of 18.5 .times. 10-5 to 19.9 .times. 10-5 m as [Na+] was varied from 12.5 to 62.5 mM. The deacylation rate constant, k3, for p-guanidinobenzoyl-APC hydrolysis was also independent of [Na+], with a value of (3.8 .+-. 1.0) .times. 10-3 s-1 in the absence of Na+ or in the presence of concentrations of Na+ up to 200 mM. Identical kinetic behavior was observed when Cs+ was substituted for Na+ in the above enzymic reaction. The pre-steady-state kinetic parameters were calculated according to the same methodology as described above. The k2,max for acylation by NPGB was found to be 1.36 .+-. 1.10 s-1, the Km for Cs+ was 12 .+-. 2 mM, the Ks for NPGB was (1.8-2.2) .times. 10-5 M, and the k3 for deacylation of p-guanidinobenzoyl-APC was determined to be (3.8 .+-. 1.0) .times. 10-3 s-1.

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