d-Alanine Carboxypeptidase from Bacillus subtilis Membranes

The purified D-alanine carboxypeptidase isolated from Bacillus subtilis was irreversibly inactivated by penicillin G and other /3-lactam antibiotics. The reaction involved an initial reversible binding step followed by an irreversible step. Both the reversible binding constant (KI) and the rate constant for irreversible inactivation (k3) were determined for a number of /3-lactam antibiotics. In general KI varied among these antibiotics much more than ka. Significant differences were also observed in the KI for the pure soluble enzyme and that for the membrane bound enzyme; these differences might be attributable to the solubility of the /3-lactam antibiotic in the membrane phase.


SUMMARY
The purified D-alanine carboxypeptidase isolated from Bacillus subtilis was irreversibly inactivated by penicillin G and other /3-lactam antibiotics.
The reaction involved an initial reversible binding step followed by an irreversible step. Both the reversible binding constant (KI) and the rate constant for irreversible inactivation (k3) were determined for a number of /3-lactam antibiotics.
In general KI varied among these antibiotics much more than ka. Significant differences were also observed in the KI for the pure soluble enzyme and that for the membrane bound enzyme; these differences might be attributable to the solubility of the /3-lactam antibiotic in the membrane phase.
Current understanding of the interaction of penicillin with the microbial cell indicates that it reacts with multiple components on the cell surface, of which one, or at least a limited number, are involved in lethality (1,2). Presumably one of these components, in Escherichia coti at least, is the transpeptidase which catalyzes the cross-linking of peptidoglycan subunits in the bacterial cell wall and is irreversibly inactivated by penicillins and cephalosporins.
Killing results from an increased sensitivity to lysis due to failure of synthesis of these cross-bridges.
It has been hypothesized that penicillin G inhibits this reaction by acting as an analog of the D-alanyl-D-alanine terminus of the peptidoglycan subunits (GlcNAc-MurNAc-L-Ala-D-Glu-L-lys-n-Ala-D-Ala) which are the substrates in this reaction (1).
Bacillus subtilis membranes contain a n-alanine carboxypeptidase which is also irreversibly inactivated by penicillins and cephalosporins (3). The function of this enzyme is unknown although it is believed to be involved in some step of cell wall biosynthesis.
The enzyme is the major penicillin binding component of B. subtilis but its inactivation is not lethal to the cell (4). The enzyme has been solubilized by the nonionic detergent *This work was supported by research grants from United States Public Health Service (AI-09152 and m-13230) and National Science Foundation (GB-29747X). This is Paper XXXIV in the series "Biosynthesis of the Peptidoglycan of Bacterial Cell Walls." $ Present address, Department of Biology, McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218.
$ To whom reprint requests should be addressed.
Triton X-100 and purified to homogeneity (5). The interaction of the purified enzyme with penicillins and cephalosporins has been studied in the present work.

MATERIALS AND METHODS
The n-ala&e carboxypeptidase was prepared and assayed as previously described (5). The antibiotics used were kind gifts of the Squibb Institute for Medical Research, New Brunswick, New Jersey; Bristol Laboratories, Syracuse, New York; and Eli Lilly and Co., Indianapolis.

Irreversibility
of Inactivation-Preincubation of the purified soluble D-alanine carboxypeptidase with penicillin G resulted in inactivation that could not be reversed by penicillinase (Table I) or excess substrate (Fig. 1). Radioactive penicillin G remained bound to the enzyme even after the enzyme was denatured in sodium dodecyl sulfate (5,6).
Titration of Enzyme with Penicillin-Titration of the enzyme with penicillin G indicated that 1 mole of penicillin was sufficient to inactivate 1 mole of enzyme (Fig. 2). E$ect of Penicillin on Enzyme Suljhydryl Groups-The n-alanine carboxypeptidase was inhibited by reagents which react with sulfhydryl groups (5), suggesting an involvement of a sulfhydryl group in the catalytic site. The inactivation of the enzyme by penicillin G involved the loss of one of the four sulfhydryl groups in the enzyme which are titrated by 5,5'dithiobis(2-nitrobenzoic acid) (Fig. 3). These data suggest that penicillin G inactivates the enzyme by reacting with a single sulfhydryl group in the active site of the enzyme, or at least that it reacts in the neighborhood of a sulfhydryl group, thereby protecting it from titration.
Determination of Kr and &--Since the carboxypeptidase is inactivated by the penicillin, the mechanism of inhibition which would be predicted is as follows where I is a penicillin, EI is a reversible complex, and EI' is the inactivated enzyme. An analysis of this type of inhibition has been presented by Kitz and Wilson (7) in considering the irreversible inactivation of acetylcholinesterase by esters of methane sulfonic acid. The same kinetic analysis has been applied in the present case. It predicts that ln(e/EO) = -k3t/ (1 + KI/I) where E is the remaining active enzyme (E + El) obtained by assay after dilution and treatment with penicillinase and E" is total enzyme. When I is constant during the reaction (i.e. Z > EO), ln(e/EO) = -k,,, t where kapp = k,/(l + KI/Z) and l/k,,, = (l/k,) + (KI/kS) x l/I. When -log e/E0 was plotted against t at different concentrations of Z, straight lines were obtained (Fig. 4) as predicted by this mechanism. The slopes of these lines give kapp as a function of Z. A double reciprocal plot of l/k,,, versus l/Z also gave a straight line (Fig.  . The intercept is then l/k, and the slope Kr/ks from which ka and Kr can be calculated. Such data obtained for propicillin are illustrated in Figs. 4 and 5 and for 10 p-lactam antibiotics are summarized in Table II. Penicillin G and penicillin V inactivation rates (k3) could not be measured at 37", but were slow enough at 4" to allow measurement.
The rate constants, ka, for the other P-lactam antibiotics were quite similar, ranging between 4.2 X 10-l s-l for propicillin and 0.2 x 10-l s-1 for cloxacillin, a variation of only 20-fold. The dissociation constants varied from 0.1 x lop3 M for penicillin G (at 4") to 28 X lOpa M for cephalothin, a variation of almost 300-fold.
Znhibition of Membrane-bound Enzyme by /I-Lactam Antibiotics-when the constants KI and kS were determined for the membrane-bound enzyme a significant difference was found in the slope of the double reciprocal plot, but not in the intercept (Fig. 5). The membrane-bound enzyme had an increased affinity for the antibiotics (Table II). This alteration in Kr for antibiotic was not reflected in an altered K, for UDP-MurNAc-pentapeptide between the membrane-bound and soluble enzyme, nor by an altered Kl for inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) (5). Thus, it appeared that the active site of the enzyme was not significantly altered during solubilization and purification. Therefore the increased affinity for P-lactam antibiotics of the membrane-bound enzyme was an environmental effect of the membrane. One possibility u-as that the hydrophobic antibiotics were "dissolved" in the mem-  (1 At 4"; assay at the lower temperature means the rate constants are not directly comparable, but the binding constants should not be very temperature dependent. brane, increasing the local concentration of the antibiotic near the active site of the enzyme. A correlation between the hydrophobicity of the penicillins and the increase in affinity that could be attributed to this process was found (Fig. 6). The two cephalosporins examined, cephalothin and cephalosporin C, did not fit on this curve. Since this family of antibiotics has a different structure, it may require a separate correlation curve. Conceivably its distribution in butanol-water is not proportional to its solubility in the membrane lipids in the same manner as the penicillins.
Furthermore presumably membrane hydrophobicity (8) slightly altered the sensitivity of the membrane-bound enzyme to p-lactam antibiotics (see 9, 10). The addition of Triton X-100 to the membrane preparations resulted in a decrease in the affinity close to that of the purified enzyme.
Protection of Carboxypeptidase from Penicillin G by Substrate-If the KI and k3 measured by the method of Kitz and Wilson (7) are measures of the binding of /3-lactam antibiotics and reaction at or near the substrate binding site, one would predict that high concentrations of substrate would protect enzyme against inactivation by &lactam antibiotics, i.e. substrate is a competitive inhibitor of inactivation (7). This prediction was verified. The enzyme could be protected from penicillin G by the presence of substrate during the preincubation (Fig. 7). Substrate protection also required zinc ions. This fact is compatible with the idea that zinc is required for the proper binding of substrate to enzyme (5).
Reciprocal Plots-Double reciprocal plots of the reaction rate data obtained at one time point intersected on the abscissa when plotted by the method of Lineweaver-Burk (11) (Fig. 8A) suggestive of competitive inhibition.
When the data were plotted by the method of Dixon (12), intersection at a point above the ordinate was again indicative of competitive inhibition (Fig. SB), but the plots were nonlinear.
The nonlinearity resulted from the irreversibility of the inhibition. At high levels of inhibition most of the enzyme was tied up as irreversibly inhibited complex, and the assumptions made in the usual rate scheme could no longer be valid.
A plot of the data by the method of Morri-  (13) and Henderson (14) for very tight binding inhibitors showed an increase in slope with increasing substrate concentration, again indicative of competitive inhibition (Fig. NY). In all cases the apparent KI measured was 1.2 to 1.7 x 1OP for penicillin G. This value is far from the true KI as measured above. Thus, misleading data can be obtained if the actual mechanism of the inhibition is not considered in the kinetic analysis.
A Hill plot (15) made at several concentrations of substrate resulted in a Hill coefficient of 1.2 to 1.3 indicating, as expected, the absence of cooperativity (Fig. 80).

DISCUSSION
The n-alanine carboxypeptidase of B. subtilis was irreversibly inhibited by penicillins and cephalosporins.
The inhibition involved two steps, a reversible binding to form a Michaelis complex, and then the irreversible reaction involving the loss of a single titratable sulfhydryl residue.
The initial binding was competitive with the substrate, and the enzyme would be protected from inhibition by the presence of substrate suggesting that penicillin, in all likelihood, reacts at the active center.
The irreversibility of the inactivation of the B. subtilis Dalanine carboxypeptidase contrasts with the reversible inhibition by penicillin G. A, the data were plotted by the double reciprocal method of Lineweaver and Burk (11). The curves intersected on the abscissa for penicillin G (data shown) and other p-lactam antibiotics (not shown). The point of intersection of the lines on the abscissa is magnified in the inset. B, a plot of the data according to the Dixon method (12) resulted in curves intersecting above the ordinate with an estimated Kr of 1.2 X 10-e M suggestive of competitive inhibition, although the extrapolation was uncertain. C, the data were reploted according to the equation I/[1 -(Vi/ Vo)] = Etotsl + K~[Stots~ + K.)/ s tatsi] (Vo/Vi).
It has been suggested that the inhibition of the Streptomyces n-alanine carboxypeptidase might be allosteric (i.e. due to binding of penicillin at a site other than the substrate binding site) because one of these enzymes shows peculiar inhibition kinetics (18). All of the dat.a obtained with the B. subtilis enzyme including the kinetics of inhibition and the protection by substrate from inactivation are compatible with the simpler hypothesis that penicillin reacts with this enzyme at the substrate binding site.
Three features are required in a /I-lactam antibiotic in order to inhibit the in situ enzyme effectively. The more effective antibiotics would have a high rate of inactivation (k3), a tight binding to the enzyme (Kr), and a high solubility in the membrane phase. Of these, the most important would seem to be the binding constant.
The side chain of the P-lactam antibiotic plays an important role in the binding.
The importance of the solubility of the penicillin in organic phases has been discussed by several authors (10,20,21) in relation to supposed increased permeability through the layers of the wall to the membrane.
However, solubility in the membrane appears to be an important feature also.