Enzymes lactamase, action

From structure-activity correlations, it has been suggested that stability to p-lactamase action is conferred through steric hindrance by the 7a-methoxyl group at the enzyme active site (Birnbaum et al., 1978). Introduction of a 7a-methoxyl group depresses the rate of enzymatic hydrolysis by E. coll TEM -lactamase by a factor of 3 x 10" for cefoxitin relative to cephalothin (Fisher and Knowles, 1978). However, work from Knowles group suggests that the effects of the methoxyl substituent extend beyond a simple reduction in as the characteristics of the hydrolytic pathway are changed (see Fisher and Knowles, 1978). 

The reaction is partly diffusion controlled and the rate constants are such that there is no single RDS for jS-lactamase action (Table 8.4), a sign of fully efficient enzymes with good substrates. Another class A enzyme, /3-lactamase I of B. cereus, has rate constants for benzylpenicillin similar to those of TEM jS-lactamase and S. aureus /8-lactamase PCI (29,30). With cephalosporin C, which is a poor substrate, is substantially larger than ki, indicating that the ring-opening step is RDS (31). 

According to their genetic relationship and their biochemical mechanism of action (3-lactamases are divided into enzymes of the serine-protease type containing an active-site serine (molecular class A, C, and D enzymes) and those of the metallo-protease type (molecular class B enzymes), which contain a complex bound zinc ion. 

An additional disadvantage with many penicillin and cephalosporin antibiotics is that bacteria have developed resistance to the drugs by producing enzymes capable of hydrolysing the P-lactam ring these enzymes are called P-lactamases. This type of resistance still poses serious problems. Indeed, methicillin is no longer used, and antibiotic-resistant strains of the most common infective bacterium Staphylococcus aureus are commonly referred to as MRSA (methicillin-resistant Staphylococcus aureus). The action of P-lactamase enzymes resembles simple base hydrolysis of an amide. 

Note that penicillins and structurally related antibiotics are frequently deactivated by the action of bacterial -lactamase enzymes. These enzymes also contain a serine residue in the active site, and this is the nucleophile that attacks and cleaves the P-lactam ring (see Box 7.20). The P-lactam (amide) linkage is hydrolysed, and then the inactivated penicillin derivative is released from the enzyme by further hydrolysis of the ester linkage, restoring the functional enzyme. The mode of action of these enzymes thus closely resembles that of the serine proteases there is further discussion in Box 7.20. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

Efforts to overcome the actions of the p-lactamases have led to the development of such p-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam. They are called suicide inhibitors because they permanently bind when they inactivate p-lactamases. Among the p-lactamase inhibitors, only clavulanic acid is available for oral use. Chemical inhibition of p-lactamases, however, is not a permanent solution to antibiotic resistance, since some p-lactamases are resistant to clavulanic acid, tazobactam, or sulbactam. Enzymes resistant to clavulanic acid include the cephalosporinases produced by Citrobacter spp., Enterobacter spp., and Pseudomonas aeruginosa. 

These substances resemble 3-lactam molecules (Figure 43-7) but they have very weak antibacterial action. They are potent inhibitors of many but not all bacterial 3 lactamases and can protect hydrolyzable penicillins from inactivation by these enzymes. -Lactamase inhibitors are most active against Ambler class A lactamases (plasmid-encoded transposable element [ ] lactamases in particular), such as those produced by staphylococci, H influenzae, N gonorrhoeae, salmonella, shigella, E coli, and pneumoniae. They are not good inhibitors of class lactamases, which typically are chromosomally encoded and inducible, produced by enterobacter, citrobacter, serratia, and pseudomonas, but they do inhibit chromosomal 3 lactamases of bacteroides and moraxella. 

All -lactam antibiotics are bactericidal. They interfere with the synthesis of the bacterial wall by inhibiting the bacterial ftanspeptidase enzymes essential for the construction of peptidoglycan of the wall. Some -lactams may be inactivated by the -lactamases (penicillinases) produced by bacteria and, thus, the activity of both penicillins and cephalosporins can be determined by their ability to withstand the destructive action of -lactamases also produced by the organism for its optimal protection. Bacterial resistance caused by -lactamase production... 

Both the cephalosporins and the penicillins owe their antibacterial action to their ability to block bacterial cell-wall biosynthesis. Cephalosporin C is less active than the penicillins, but is less susceptible to enzymatic destruction by /3-lactamases, which are enzymes that cleave the lactam ring. In fact, the so-called resistance of staph bacteria to penicillins is attributed to the propagation of strains that produce /3-lactamase. Numerous semisynthetic penicillins and cephalosporins have been made in the hope of finding new broad-spectrum antibiotics with high activity but with greater /3-lactam stability. Several of these are in clinical use. 

The action mechanism of a novel class of monobactams, inhibitors for the class A (3-lactamases has been reported in 1999 and is showed in Scheme 103 [310-313]. As exemplified by structure I, the inhibitor acylated rapidly the active site serine of (3-lactamase and the tosylate was released from species II. The acyl-enzyme underwent fragmentation, resulting in enzyme inhibition by formation of three distinct products, depending on the type of functionality linked to the inhibitor (III, IV, or V, Scheme 115). 

It is known that p-lactamase catalyzes the rapid hydrolysis of the p-lactam ring of penicillins and cepharosporines. The hydrolytic activity of these enzymes eliminates the bacteriocidal action of many p-lactam antibiotics and makes the organism resistant to these molecules. For this reason, the p-lactamase inhibitors have long been regarded as promising targets from a medicinal viewpoint. A comparison between the kinetic characteristics of p-lactamase and penicillin-sensitive enzymes (carboxy-peptidase and transpeptidase) is of interest in this respect. p-Lactamases very efficiently hydrolyze p-lactam in contrast to penicillin-sensitive enzymes [high /e4 in Eq. (9)]. 

Enzyme inhibitors such as cloxacillin and methicillin have been shown to potentiate the action of certain penicillins and cephalosporins against Ps. aeruginosa Figure 7.7). Thus, the presence of cloxacillin, which is a strong inhibitor of the inducible enzyme, potentiates the effect of cephaloridine which alone is susceptible to hydrolysis by the pseudomonas lactamase. Cloxacillin shows no antibacterial activity against... 

The class C >8-lactamases include the chromosomal cephalosporinases of Gram-negative bacteria. They are large molecules of approximately 39,000 Da and show no sequence homology with the other classes. The amino-acid residue directly implicated in enzyme action of this class has been identified as serine-80. 

The second approach to combat the action of 8-lactamases was to identify an agent that was capable of inhibiting the bacterial enzyme, and in doing so to protect the yS-lactam antibiotic from destruction. 

Clavulanic acid is also a mechanism-based inhibitor and its mode of action is believed to involve ring opening of the initially formed acyl-enzyme complex (18) to the keto-derivative (19), which may then tautomerise to the hydrolytically more stable -amino-acrylate (20) Scheme 6.4). This transiently inhibited form may hydrolyse to re-release active enzyme or react further with the enzyme to produce irreversibly inhibited forms. It has been shown that approximately 115 molecules of clavulanic acid are destroyed per molecule of enzyme before the j8-lactamase is irreversibly inactivated. Whilst irreversibly inactivated forms are known to exist, the nature of these products is not yet known. Possible structures are (21) and... 

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