Serine residues bacterial

P-Lactam antibiotics exert their antibacterial effects via acylation of a serine residue at the active site of the bacterial transpeptidases. Critical to this mechanism of action is a reactive P-lactam ring having a proximate anionic charge that is necessary for positioning the ring within the substrate binding cleft (24). 

Several drugs in current medical use are mechanism-based enzyme inactivators. Eor example, the antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidoglycan chains during synthesis of bacterial cell walls (Eigure 14.17). Once cell wall synthesis is blocked, the bacterial cells are very susceptible to rupture by osmotic lysis, and bacterial growth is halted. 

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

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. 

The site of action in the 3-lactam antibiotics is muramoylpentapeptide carboxypeptidase, an enzyme that is essential for cross-linking of bacterial cell walls. The antibiotic resembles the substrate of this enzyme (a peptide with the C-terminal sequence D-Ala-D-Ala) and is therefore reversibly bound in the active center. This brings the 3-lactam ring into proximity with an essential serine residue of the enzyme. Nucleophilic substitution then results in the formation of a stable covalent bond between the enzyme and the inhibitor, blocking the active center (see p. 96). In dividing bacteria, the loss of activity of the enzyme leads to the formation of unstable cell walls and eventually death. 

The action of p-lactam antibiotics is considered to be due to the formation of an acyl enzyme with carboxypeptidases and transpeptidases which are involved in the biosynthesis of bacterial cell walls38). A three-step mechanism involving a stable acyl-enzyme intermediate (El ), a participating active site serine residue, and a very slow decay process (k4.) was proposed [Eq. (9)]59). 

These /3-lactam antibiotics apparently work by interfering with the synthesis of bacterial cell walls. Figure 21-11 shows how the carbonyl group of the /3-lactam acy-lates a hydroxyl group (from a serine residue) on one of the enzymes involved in making the cell wall. The acylated enzyme is inactive for synthesis of the cell wall protein. This acylation step is unusual because it converts an amide to an ester, an uphill reaction that we would assume to be endothermic. With this /3-lactam, however, the strain of the four-membered ring activates the amide enough for it to acylate an alcohol to form an ester in an exothermic step. 

Penicillin imitates D-AIa and binds to the active site of the enzyme, encouraging the OH group of a serine residue to attack the reactive, strained [5-lactam. This same OH group of the same serine residue would normally be the catalyst for the D-Ala-D-Ala cleavage used in the building of the bacterial cell wall. The reaction with penicillin protects the serine and irreversibly inhibits the enzyme. The bacterial cell walls cannot be completed, and the bacterial cells literally burst under the pressure of their contents. Penicillin does not kill bacteria whose cell walls are already complete but it does prevent new bacteria being formed. 

SR-proteins can be expressed as recombinant proteins in Escherichia coli, but they lack the post-translational phosphorylation of the serine residues and are poorly soluble in the absence of chaotropic reagents. It is possible to phosphorylate bacterially produced protein by preincubation in nuclear extract or, even more efficiently, by the addition of purified recombinant SR-protein specific kinases Clk/Sty8 or SRPK.9 Soluble and phosporylated SR-proteins can also be produced in insect cells using the baculovirus system10 although it is not resolved whether these proteins behave exactly as those produced in human cells. 

Boronic acid derivatives have been shown to inhibit -lactamases [168]. The boronic acid function, which is isoelectronic with a protonated carboxyl group, is presumed to block enzymatic activity by forming a stable tetrahedral borate complex with the active serine residue. Boronic acids 319, structurally related to penicillin G and methicillin, were synthesised in three steps from dibutyl iodomethaneboronate [169] (Scheme 97). They are inhibitors of P-lactamase I from B. cereus. There are presently no reports of boronic acid derivatives acting as inhibitors of bacterial D,D-peptidases. 

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