Michael Page is Professor of Chemistry in the Department of Chemical and Biological Sciences and Dean of the School of Applied Sciences, The University of Huddersfield, Queensgate, Huddersfield, United Kingdom.

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Understanding Metallo ß-Lactamases

A major role of zinc in these enzymes is to act as a Lewis acid rather than providing a nucleophile when hydrolyzing ß-lactam antibiotics

Michael I. Page

The ability to produce ß-lactamase enzymes is the major cause of the resistance of bacteria to ß-lactam antibiotics. These enzymes catalyze hydrolysis of four-membered ß-lactam rings, converting them into ß-amino acids that, unlike active members within this class of antibiotics, no longer interfere with bacterial cell synthesis and growth. The ß-lactamase enzymes are divided mechanistically into two groups, with those in classes A and C using serine as an active site residue, and those in class B using a metal ion, which is invariably zinc.

The class B metallo ß-lactamases were discovered more that 40 years ago by E. P. Abraham at the University of Oxford in a strain of Bacillus cereus that also produces two serine ß-lactamases. By 1985 researchers knew of two metallo ß-lactamases, whereas currently about 20 different strains, most of them human pathogens, are recognized as carrying such ß-lactamases. The genes encoding these enzymes generally are chromosomal but some are on plasmids, which enables them to spread among microorganisms more readily and thus makes them a greater cause of public health concern.

The metallo ß-lactamases are broad-spectrum enzymes that very effectively catalyze the hydrolysis of not only penicillins and cephalosporins but also other ß-lactam antibiotics, making pathogens that carry these enzymes a real problem in the clinic. In particular, they catalyze the hydrolysis of carbapenems, such as imipenem, that are generally resistant to serine ß-lactamases. Indeed, when these antibiotics were introduced, no known ß-lactamases catalyzed their hydrolysis. Currently, although several compounds effectively inhibit these metallo enzymes in vitro, none is clinically useful.

Metallo ß-Lactamases Being Intensively Studied

Figure 1

The primary sequences of more than 20 metallo ß-lactamases are now available, along with refined crystal structures for four of them, each of which appears capable of binding two zinc ions. Although procedures for purifying and crystallizing these enzymes might distort this picture in some cases, all these enzymes require at least one zinc per molecule of enzyme to be catalytically active.

Some investigators divide these enzymes into three subgroups—designated B1, B2, and B3—on the basis of the residues that bind the zinc ions. Among those belonging to the most common B1 type, the zinc ion in the first zinc site is in a four-coordinate tetrahedral geometry that is bound by three histidine residues, while the fourth coordination site is occupied by a water molecule. The second zinc binding site of the B1 proteins contains its metal ion in a five-coordinate arrangement with trigonal bipyramidal geometry. In this configuration, three different amino acid residues—aspartate, cysteine, and histidine—serve as ligands, furnishing oxygen, nitrogen, and sulfur atoms, respectively, to link with zinc. In addition, the zinc binds two water molecules, one which is not bound elsewhere and another which bridges with the other zinc ion. The two zinc ions thus are fairly close to one another, separated by only about 3.5 Å, particularly in those enzymes that bind these ions very tightly.

These enzymes target the carbon-nitrogen bond and open the four-membered ring that is characteristic of ß-lactam antibiotics. In the first step of this hydrolytic reaction, a new bond forms between the ß-lactam carbonyl carbon and oxygen, which comes from water, assumed to be the one bound to zinc. In addition to breaking the carbon-nitrogen bond, various proton transfers occur. Metal ions can increase the rates of both steps—the first in which a new carbon-oxygen bond forms, and the second in which the carbon-nitrogen bond of the ß-lactam ring breaks. Despite a misplaced emphasis on that first step, breaking the carbon-nitrogen bond in peptides and, surprisingly, in ß-lactams is not easy and also requires catalysis.

The zinc cation, with its high positive charge density, could play a key role in this step, acting in one of several ways. For one, this charge density reduces the pKa of the water that is bound to the zinc ion, forming the rough equivalent of a zinc-bound "hydroxide ion."If this pKa were lowered to 7, then the acidity of this bound water molecule approaches that of dihydrogen phosphate ion or MOPS, suggesting that this zinc-bound "hydroxide ion" would be no more nucleophilic than either of these bases. Alternatively, the zinc ion might act as a Lewis acid, polarizing the carbonyl group while stabilizing the oxyanion that is formed following nucleophilic addition to the carbonyl group of the ß-lactam. Yet another possibility is that the zinc ion facilitates carbon-nitrogen bond fission in the ß-lactam ring by directly coordinating to the nitrogen and stabilizing the nitrogen anion that is expelled. Finally, the water molecule bound to the zinc could act as a general acid catalyst, donating a proton to that nitrogen to facilitate carbon-nitrogen bond fission.

Several Reasons Why Bacteria Use Zinc in Metallo-Enzymes

Figure 2

Why do bacteria produce a zinc metallo enzyme? One important requirement is the protein's ability to sequester particular metal ions from the environment. Ions are selected primarily on the basis of size, charge, and "hardness," which is a measure of an ion's relative capacity to combine with oxygen, sulfur, and nitrogen ligands. One of the interesting things about zinc is that it is on the borderline in hardness and it therefore combines equally well with oxygen, sulfur, and nitrogen ligands and, in the case of one of the classes of ß-lactamases, the metal binds all three of these ligands. Another important feature of zinc is that although its geometry is predominantly a four-coordinate tetrahedral, it can change very readily to five- or six-coordination with little expense of energy. Finally, zinc ions have a d10 electronic configuration, meaning there is no redox chemistry and avoiding ligand field stabilization energy and any complications that would occur by changing the number and nature of ligands on the zinc.

All these properties make zinc the favored metal for hydrolytic catalytic activity. To make this enzyme, bacteria have to sequester zinc from the environment, which is not easy. The hydration energy of zinc ions is very high—at least 500 kilocalories per mole—making zinc extraction and binding a very energetically expensive process.

Several changes occur when zinc is taken out of the environment. In aqueous solution, zinc is generally six coordinate with an octahedral geometry, whereas in the protein it is generally tetrahedral. Hence, on binding, the ligands change, the geometry of their binding changes, and the pKa of the water that is bound to the zinc also changes. Water has a pKa of about 16. However, when it is bound to zinc, the pKa decreases tremendously to around 9, making the water much more acidic. When zinc with its bound water molecule is coordinated to a protein, typically the pKa is reduced even further, down to about 6 or 8. The main reason for this change in pKa is the altered coordination chemistry for zinc from an octahedral to a four-coordinate system, which increases the positive charge density on the cation and makes the bound water molecule more acidic. This effect of charge density is important when looking at mutants where the ligands in the enzyme bound to the metal-ion are changed, because of the effect that these changes may have on the pKa of the zinc bound water. For example, if a neutral histidine ligand of zinc is replaced by negatively charged aspartate or cysteine, then the pKa is increased by about 2 units.

Investigating the Detailed Mechanism of the Metallo ß-Lactamases

The mechanism of the metallo ß-lactamases may well not be unique but may vary for different substrates and with the source of the enzyme. For example, our studies with the Bacillus cereus metallo ß-lactamase show that, by increasing the zinc ion concentration 100,000-fold, the catalytic activity of the mono form of the zinc enzyme changes by less than a factor of 2. Thus, although the enzyme can bind two zinc ions, our kinetic evidence suggests that the second zinc does not have a significant catalytic effect.

One of the common features of hydrolytic metallo enzymes, particularly metallo proteases such as carboxypeptidase, is uncertainty over mechanism. For example, there is often an ionized amino acid—commonly aspartate—in the active site that might act as a nucleophile to form an anhydride intermediate which, in principle, could be trapped with methanol to form an ester. However, with the metallo ß-lactamases, no methyl ester forms and adding methanol does not affect the rate of hydrolysis. Such experiments, although not ironclad, indicate that the ß-lactamase mechanism is not nucleophilic.

The pH dependence of the rate of hydrolysis of benzyl penicillin catalyzed by the metallo enzyme reveals several potentially important features. First, the catalytic activity is pH independent over a wide range, with a pH bell profile that is typical of two ionizations with pKa's of about 6 and 9. Moreover, changing the pH in the acid region by 1 unit changes catalytic activity 100-fold, suggesting that two groups are ionizing. The enzyme-catalyzed hydrolysis likely involves at least three bases and a proton.

The pKa of the zinc-bound water could represent either of the ionizations indicated at pH 6 or 9 but which can be identified from the pH dependence of the inhibition constants of some thiol inhibitors. We made a series of thiol inhibitors that bind to the zinc ion, and their inhibition constants show a similar pH independence to that for the hydrolysis reaction, indicating that the water bound to zinc is ionized at neutral pH and confirming that the pKa of the water bound to zinc is 5.7. There are two catalytically important groups of about pKa 6 in this enzyme, one of which is the water bound to zinc and the other is likely the carboxylate group of the active site aspartate. An important feature of having two basic groups is that they are both anions, meaning the transition state is dianionic. This feature may prove a useful indicator for designing inhibitors of the zinc enzymes.

Zinc Ions in ß-Lactamases Likely Act in Dual Role when Hydrolyzing Antibiotics

We believe that the zinc ions in metallo enzymes act in a dual role during hydrolysis of ß-lactam antibiotics—providing, first, a Lewis acid to polarize the carbonyl group and, second, a "hydroxide ion" bound to zinc to act as a nucleophile to attack the carbonyl group to form a tetrahedral intermediate. Because the zinc-bound hydroxide is only a weak nucleophile, this step is reversible. The role of the aspartate is to deprotonate the hydroxyl group in the intermediate to form a dianion. Because the aspartate then exists in its neutral acidic form, it can act as a proton donor to the ß-lactam nitrogen to facilitate carbon nitrogen bond fission to form the end products.

In the ß-lactamase enzymes that contain two zinc ions, the bridging ion might be acting as the immediate nucleophile during the hydrolytic step. In such enzymes, the zinc-bound "hydroxide ion" is an extremely weak nucleophile, meaning the first addition step to the ß-lactam would almost certainly be reversible. The other water that is coordinated to the second zinc can act as a general acid, a proton donor to facilitate carbon nitrogen bond fission to form the products. There is an intermediate formed in these reactions, at least with nitrocefin as a substrate, and these findings suggest that the zinc ion coordinates directly to the ß-lactam nitrogen and thereby stabilizes nitrogen anion expulsion, which is subsequently protonated, according to Steve Benkovic at Pennsylvania State University in University Park, Pa., and his collaborators.

Site-directed mutagenesis of the mono-zinc form of the ß-lactamase from B. cereus indicates that the pKa of the zinc-bound water can be increased by making the zinc effectively less positive due to a change in ligands around the metal ion. A higher pKa reduces the concentration of the zinc-bound hydroxide ion at pH values below the pKa but, at the same time, makes it a better nucleophile. However, because the zinc has less positive charge density, these changes will decrease the ability of zinc to stabilize the oxygen anion and polarize the ß-lactam carbonyl.

If the zinc is acting mainly as a Lewis acid stabilizing the tetrahedral intermediate, then one would expect the mutant to show reduced activity, whereas if its main role is to provide a water-bound nucleophile, then activity would be expected to increase. Replacing one of the zinc-bound histidines by serine, in fact, reduces the activity of the enzyme. The pH dependence of the rate of hydrolysis of ß-lactams catalyzed by the mutant now shows two separate ionizations on the acid limb. With serine replacing histidine, the positive charge density on the zinc is decreased, thereby increasing the pKa of the zinc-bound water to 7.3, separating it from the aspartate. The decreased activity of this particular mutant demonstrates that one of the major roles of zinc is to act as a Lewis acid rather than to provide the nucleophile. By decreasing the positive charge density on zinc and increasing the pKa of the zinc-bound water, the ability of the zinc to stabilize the developing negative charge density on the ß-lactam carbonyl oxygen is decreased.

Because one of the important functions of zinc is to polarize the ß-lactam carbonyl group, we made a series of cysteinyl dipeptide inhibitors of these enzymes in each of which the thiol anion can coordinate to the zinc ion. Hydrophobic residues increase the ability of the zinc enzyme to bind these inhibitors. Among a series of diastereoisomers, the DD diastereoisomer proved a better inhibitor than the others. In penicillins and cephalosporins, the acyl amino side chain has an l stereochemistry at C6/7 and a D stereochemistry at the C3/4 bearing carboxylate, respectively. If the cysteinyl dipeptides are bound to the zinc, the stereochemistry has to be inverted to enable the best molecular recognition of the other substituents.

SUGGESTED READING

Bounaga, S., A. P. Laws, M. Galleni, and M. I. Page. 1998. The mechanism of catalysis and the inhibition of Bacillus cereus zinc-dependent ß-lactamase Biochem. J. 331:703-711.

Bounaga, S., M. Galleni, A. P. Laws, and M. I. Page. 2001. Cysteinyl peptide inhibitors of B. cereus zinc ß-lactamase. Bioorg. Med. Chem. 9:503-510.

de Seny, D., C. Prosperi-Meys, C. Bebrone, G. M. Rossolini, M. I. Page, P. Noel, J.-M. Frere, and M. Galleni. Mutational analysis of the two zinc binding sites of the Bacillus cereus 569/J/9 metallo-ß-lactamase. Biochem. J., in press.

Page, M. I., and A. P. Laws. 1998. The mechanism of catalysis and the inhibition of ß-lactamases. Chem. Commun. 1998:1609-1617.

Paul-Soto, R, R. Bauer, J.-M. Frere, M. Galleni, W. Meyer-Klaucke, H. Nolting, G. M. Rossolini, D. de Seny, M. Hernandez-Valladares, M. Zeppezauer, and H.-W. Adolph. 1999. Mono- and binuclear Zn²⁺- ß-lactamase J. Biol. Chem. 274:13242-13249.