Kinetic parameters are reported for the Bacillus cereus P-lactamase Iand P-lactamase II -catalysed hydrolysis of a series of thirty-seven cephalosporins substituted in the 7-position. These are compared with the second-order rate constants for the hydroxide ion-catalysed hydrolysis of these derivatives. There is no significant dependence of the rate of the base-catalysed hydrolysis upon the nature of the side-chain substituent. For P-lactamase I, kc,,/K, varies over 2 x lo5 but for plactamase II the variation with substituents is only 10. For alkyl substituents, k,,JK, increases with chain length and passes through a maximum, for p-lactamase I this is with the undecyl derivative and for P-lactamase II the octylcephalosporin. For p-lactamase I, but not for P-lactamase II, the tbutylcephalosporin is a very poor substrate. There is no evidence for a significant cavity in either enzyme to host aromatic residues. An ionised carboxylate residue on the side-chain significantly reduces reactivity with P-lactamase I but not p-lactamase II. It is suggested that a carboxy group on P-lactamase I acts as a general catalyst facilitating p-lactam C-N bond fission.The two major classes of p-lactam antibiotics are the penicillins (1) and the cephalosporins (2).t Bacteria which are normally susceptible to these antibiotics may be rendered insusceptible if they can produce P-lactamases, enzymes that catalyse the hydrolysis of the p-lactam.There are many different p-lactamases which have been classified on the basis of substrate profile (the ability to catalyse the hydrolysis of various p-lactam antibiotics e.g. penicillinase or cephalosporinase), on the basis of their sequence or on a mechanistic bask2 For example, the TEM2 P-lactamase from E. coli is very efficient and one molecule of the enzyme can catalyse the hydrolysis of 2 OOO molecules of benzylpenicillin in 1 ~e c o n d . ~ However, this enzyme is more than loo00 less reactive towards some cephalo~porins.~ Conversely, one molecule of the P99 P-lactamase can hydrolyse 2 OOO molecules of cephaloridine in 1 second. One mechanistic class comprises plactamases that are serine enzymes; this is subdivided into classes A and C based on the amino acid sequences.2 The other mechanistic class of p-lactamases is distinguished by the requirement for zinc(r1) ions and a lack of sequence homology to enzymes of the other classes. There are two well characterised but unrelated enzymes of this class B-p-lactamase I1 from B. cereus and p-lactamase L-1 from Pseudonomas maltophilia. RCONH RCONH An example of the systematic nomenclature system for the trivially named cephalosporins, includes 3-[(acetyloxy)methyl]-8-oxo-7-(2-pheny1acetamido)-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid for benzylcephalosporin.
a-Methyl (3Sf5Rf6R)benzylpenicilloate is a reversible competitive inhibitor of (3-lactamase I from Bacillus cereus; the inhibition constant Ki is 6.38 x l o -4 ~ and is independent of pH between pH 5 and 8 which is compatible with binding of the free base form of the thiazolidine residue to the enzyme.
Second-order rate constants for the alkaline hydrolysis of 3-thiol substituted cephalosporins are independent of the pK, of the thiol over a pK, range of 9. If there is a leaving group at C-3' it is expelled after the p-lactam ring is opened and the expulsion of the leaving group does not enhance the rate of p-lactam C-N bond fission. The zinc enzyme P-lactamase II is about a 100-fold better catalyst than the serine enzyme P-lactamase I for the hydrolysis of the same cephalosporin. The second-order rate constant kc,,/K, for both P-lactamase enzymes shows n o dependence on the nature of the substituent at C-3' which is not explicable by the different chemical reactivity of thecephalosporins. There is no evidence for a significant recognition site in either enzyme for the C-3' substituent. The kinetic parameters kcat and K, for the p-lactamase I-catalysed hydrolysis may be complicated b y the formation of intermediates.
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