Mi. Page et al., PH-DEPENDENCE OF AND KINETIC SOLVENT ISOTOPE EFFECTS ON THE METHANOLYSIS AND HYDROLYSIS OF BETA-LACTAMS CATALYZED BY CLASS-C BETA-LACTAMASE, Journal of the American Chemical Society, 117(49), 1995, pp. 12092-12095
The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloa
cae P99 class C beta-lactamase and the pH dependence of k(cat) indicat
es that the catalytic groups involved of pK(a) ca. 5 and 10 are the sa
me as those for hydrolysis. The kinetic solvent isotope effect (KSIE)
k(cat)(H)2(O)/k(cat)(D)2(O) is 1.42 for both the hydrolysis and methan
olysis of benzylpenicillin. However, there is an inverse KSIE on k(cat
)/k(m) of 0.83 +/- 0.05 for the hydrolysis of benzylpenicillin and cep
haloridine. There is also an abnormally high shift in the low pK, on g
oing from H2O to D2O of 0.85 +/- 0.15 from the pH dependence of both k
(cat) and k(cat)/K-m for both methanolysis and hydrolysis. The D2O shi
ft on the high pK(a) of ca. 10 is the normal value of ca. 0.4. These r
esults are consistent with a strongly hydrogen bonded system acting as
the general base catalyst and low fractionation factors for the hydro
gens involved. It is probable that this represents the tyrosine phenox
ide ion hydrogen bonded to two lysine ammonium ions.