The pH-dependence and group modification of β-lactamase I

Abstract

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.

Protonation of the β-lactam nitrogen is the trigger event in the catalytic action of class A β-lactamases

“…The pH profiles of the steady-state kinetic parameters k cat ͞K M and k cat are approximately bell-shaped with ratecontrolling ionization constants pK a1 and pK a2 centered on values of Ϸ 4.4 and Ϸ 8.2, respectively (8)(9)(10)(11)(12). It has been suggested that K73 is uncharged in TEM-1 ␤-lactamase and functions as the proton acceptor (4,13).…”

Bacillus cereus 569/H β-lactamase I: Structure-function relationship

“…Previous studies had demonstrated rapid inactivation of ~-lactamase I in the presence of a water soluble carbodiimide (37). In the current investigation, taurine was used to convert the reactive O-acylisourea intermediate (formed during the reaction of DCC with carboxyls) to a stable amide derivative.…”

“…Earlier attempts using enzymatic and chemical methods have failed to reveal any C-terminal residue in ~-lactamase I (35,37). The broad specificity of carboxypeptidase Y as well as its resourcefulness as an agent in the elucidation of the amino acid sequence of the C-terminal region of proteins (17,26) prompted us to reinvestigate the nature of the C-terminal residue of ~-lactamase I.…”

“…Conversion of the four histidine residues of [~-lactamase of B. cereus 569/H to the corresponding N-carbethoxy derivative has been accomplished with no effect on enzymatic activity (12,9). Reaction of the enzyme with carbodiimides and other carboxyl specific reagents has been found to effect inactivation (37). Hitherto, no efforts have been directed towards the assessment of the role played by either the amino or the guanidino functions present in the enzyme.…”

Design of enzyme‐pH electrodes

“…The design as well as the kinetic parameters used in computing curve b in Figure 5 and the profiles in Figure 6b are those corresponding to the penicillinase-pH electrode developed by Nilsson et al (1973) for monitoring penicillin-G. From the thickness of the membrane used in their experiments, the enzyme loading of their membrane, and the kinetic parameters of penicillinase (Waley, 1975) we computed that the experimental electrode of Nilsson et al had a Thiele modulus of 18.8. In their experiments, Nilsson et al maintained the bulk solution a t the optimal pH of the enzyme and investigated the electrode response in the presence of 5 x lo-, M sodium phosphate buffer for bulk substrate concentrations ranging from 5 x to 1 x M. As was already noticed from Figure 6b, under these conditions f + 1 for the entire range of substrate concentrations investigated by these authors.…”