TABLA 2.2.11 INMIGRANTES Y EMIGRANTES INTERIORES DE VALLADOLID 1998-

The role of serine in the active sites of enzymes has been best studied for the serine proteases. Similarly to the p-lactamases, the serine proteases catalyse the hydrolysis o f an amide bond. In chymotrypsin, one of the best understood examples of the serine proteases. Catalysis is facilitated by three key residues, through their chemical nature and orientation within the active site, known as the catalytic triad. Typically the catalytic triad consists of a histidine, an aspartate and a serine (Neurath H. 1989 and references therein). The histidine acts as a general base to abstract a proton from the serine hydroxyl side chain thereby increasing the nucleophilicity o f this serine. The third group in this triad is an acidic residue, usually aspartate, which acts to orient the histidine residue and also to neutralise the charged histidine intermediate. The mechanism involves the serine making a nucleophillic attack on the carbonyl carbon of the amide group to form an acyl enzyme. The amide bond is then cleaved by back donation of a proton from the histidine, now acting as a general acid, to the amide nitrogen. Déacylation of the enzyme then follows and involves an activated water molecule.

The catalytic machinery o f the serine proteases, however, does not always consist of the catalytic triad. Indeed with certain proteases a Ser/Lys dyad has been found to be the catalytically active group (Paetzel M et. a l, 1997). In this mechanism, the lysine is thought to act as a general base, i.e, play a similar role to the histidine in the classical catalytic triad, and abstract a proton from the active site serine. However, for lysine to do this, its side chain must be in the deprotonated state. The pKa of lysine is normally around 10.8 and therefore it is not clear how the lysine would be able to exist in its deprotonated form at phyisological pH. One possibility is that a positive charged active site may aid in the lowering of the pK of this lysine. It is also thought that hydrophobic residues in the vicinity of the active site could contribute to the lowering of the lysine’s pK. Buried lysine’s have been previously found to have p K ’s as low as 6.5 (Doa-Pin et. al, 1991). The absence of an acidic residue is crucial to the dyad mechanism. The presence of a negative charge near the residues that comprise the dyad would lead to an elevated pK of the catalytically implicated lysine thus disabling its ability to act as a general base. An alternative role of the conserved lysine however, could be to increase the local positive charge in the vicinity o f the active site serine so that its hydroxyl group is already deprotonated and thus removing the need for a general base in the acylation step (Moews P C et. a l, 1990).

The Ser/Lys dyad is present in all serine p-lactamases but the mechanism o f the enzymes of the various classes appear somewhat different. With the Class A enzymes, which contain the additional element the omega loop, there were two candidates for the role of general base, namely Glu 166 (from the omega loop), and Lys 73. A pH rate profile of this enzyme revealed that two groups were important for catalysis with pK ’s of 5.6 and 9 (Page M et. a l, 1998). The general base would be expected to correspond to the lower pK of 5.6. Subsequently, the role of Lys 73 as the general base was found to be unlikely from 13C NMR studies which showed the lysine to have a normal pK (Damblon et. a l, 1995). However, there was conflicting

evidence was found for Glu 166 as the general base. A Glu 166Asp mutant of the Bacillus cereus I enzyme (class A) showed decreased rate constants for both the acylation and déacylation steps, suggesting that this residue may playing a similar role of the histidine residue in the classic catalytic triad (Leung et. a l, 1994). However, other Glu 166 mutants have suggested that only the déacylation step is affected (Escobar et. a l, 1991). As a consequence of these findings, it is generally accepted that Glu 166 acts as a general base in the déacylation reaction, but its exact role in the acylation process remains poorly understood as is the role for Lys 73.

With the Class C p-lactamases, the absence of an equivalent residue to Glu 166 is very likely to result in a more positively charged active site. Such an environemnt may indeed be able to lower the pK of the consreved lysine so that it could act as the general base. However, superimposition of the active site of a class C enzyme (from Citrobacter jreundii) on the corresponding groups of chymotrypsin, it was observed that the oxygen group of the conserved tyrosine (in element 2) of the former was in a position equivalent to the proton accepting imidazole nitrogen of the catalytic triad His of the latter (Oefner et. al, 1990). Hence making this tyrosine a candidate for the general base. However, the normal pK of the tyrosine hydroxyl group is 10.8 and thus would have to be considerably lowered. This was suggested to occur through electrostatic affects generated by the interaction of the side chain lysine from element 1 and a second conserved lysine with the tyrosine oxygen atom via hydrogen bonds (Damblon eA a l, 1995).

In summary, the mechanism of serine based proteolytic enzymes and the serine p- lactamases are similar probably due to convergent evolution. That is, there is an absolute requirement for a serine that is activated either through a general base or due to the accumulation of positive charges at the active site. The catalytic core can be presented in the form of the classical catalytic triad or the Ser:Glu/Lys/Tyr dyads. In

the latter cases, the high normal pKa of the general base may be lowered by hydrogen bonding and hydrophobic affects. The active site provides residues that form the oxyanion hole required to stabilise the oxaynanionic tetrahedral acyl enzyme inetrmediate. In chymotrpsin, the oxyanion hole is comprised of the main chain NH- groups o f Ser 195 and Gly 193. Similar oxyanion holes have been observed in the serine p-lactamases (Ser 70 and Ala 237 in class A and Ser64 and Ser/Ala318 in Class C) and the PBP’s (Ser 62 and Thr 301 in the S. R61 DD-peptidase). The formation of the tetrahedral intermediate requires two proton transfer steps, i.e; proton removal from the attacking serine and proton donation to the departing amide nitrogen. Déacylation also involves two proton steps, i.e; removal o f a proton from a water molecule by the general base and then donation of a proton to the departing serine.

Figure 7 A schematic of the general mechanism proposed for serine p- lactamases. Oxyanion hole - o Z l I I S e rO --- & NH S e r ' " H B Oxyanion hole NH —— OH NH OH S erO HB + S erO B NH SerOH B

The general base is depicted as B. The mechanism has been discussed in detail in the text. This figure was adapted from Page et. al. 1998.