pH Dependence and solvent deuterium oxide kinetic isotope effects on Bacillus cereus .beta.-lactamase I catalyzed reactions

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Abstract
The solvent kinetic isotope effects (SKIE) on kcat (DV) and on kcat/Km [D(V/K)] were determined for the B. cereus .beta.-lactamase I catalyzed hydrolysis of 5 substrates that have values of kcat/Km varying over the range (0.014-46.3) .times. 106 M-1 s-1 and of kcat between 0.5-2019 s-1. The variation of D(V/K) was only from 1.06-1.25 among these compounds and that in DV was from 1.50-2.16. These results require that Dk1, the SKIE on the enzyme-substrate association rate constant, and D(k-1/k2), that on the partition ratio fo the ES [enzyme-substrate] complex, both be near 1. The larger SKIE observed on DV requires that an exchangeable proton be in flight for either or both the acylation and the deacylation reaction. The pH dependence of the values of kcat/Km for 3 substrates shows identical pKa of 5.5 and 8.4. This identity combined with the fact that only 1 of these 3 substrates is kinetically sticky proves that the substrates can combine productively with only 1 protonic form of the enzyme. There is considerable substrate variation in the pKa values of kcat observed vs. pH profiles; the inflection points for all substrates studied are at pH values more extreme than are observed in the pH profiles for kcat/Km.