GLOSARIO DE INDICADORES

The observation of acyl t r a n s p e p t i d a t i o n S f indicated that the 'acyl enzyme' should

exist, just as amino transpeptidation had suggested the existence of an ‘amino enzyme’ intermediate. Acyl transpeptidation involves the transfer of the amino portion of the substrate to a suitable peptide acceptor.

HgN-Z + H2N-X-CONH-Y-CO2H ^ HgN-X-CONH-Z + NgN-Y-COgH Scheme 1.3.4: Acyl transpeptidation

An acyl intermediate was proposed as the central interm ediate in acyl transpeptidation. This hypothesis was supported by the work of Akhtar et al.^^ Their results from trapping experiments using tritiated methanol and their observation of a covalently bound intermediate in the hydrolysis of Cbz-Tyr-pH]Tyr were consistent with an acyl intermediate. However, these experiments proved to be irreproducible and were later retracted.^^ The overall pathway of hydrolysis involving a covalent acyl intermediate (E-CO-O-CO-X) is outlined below in Scheme 1.3.5.

ki k2

E-CO2H + X-CQ-NH-Y— E-C02H[ X-CO-NH-Y] E-CO-O-CO-X

k_i k_2 +

Y-NH2 ^3

E-CO-O-CO-X + H2O E-CO2H + X-CO2H k-3

Scheme 1.3.5: Acyl enzyme hydrolysis where E represents the enzyme

Acyl transpeptidation occurs, instead of hydrolysis, when the acyl intermediate is intercepted by a free amino group, from either the substrate or another peptide. The mechanism of formation of the acyl intermediate (see Scheme 1.3.6 below) is much simpler than the mechanism for the amino intermediate formation.

X— c

NH-Y _X

“ • ^ 0 o

+ HgN-Y

where E represents the enzyme

Hunkapiller and Richards^® suggested that this acyl intermediate may then react with the freed amino group of HgN-Y to form the covalent amino intermediate discussed above. Hence, this intermediate was proposed to be central to both acyl and amino hydrolysis and probably therefore to the acyl and amino transpeptidation mechanisms.

Acyl transpeptidation reaction products were observed In some cases to give much higher yields than the corresponding hydrolysis, indicating that covalent (acyl) intermediates may also be required in the hydrolysis process. The experiments of Newmark and Knowles^"* on [^^C]Leu-Tyr-pH]Leu showed that both amino and acyl transpeptidation occurred simultaneously with this substrate (though acyl transpeptidation dominated). They concluded that the relative importance of acyl and amino transfer probably depended only on the ease with which the amino and acyl portions leave the active site after hydrolysis. A common "intermediate” for both hydrolysis pathways is implicit in this suggestion, possibly the acyl intermediate shown above.

As for the amino intermediate, the postulated acyl intermediate in the acyl transfer could not be trapped with nucleophiles such as hydroxylamine®® and methanol.^^^ A somewhat perplexing observation is the lack of either kind of transpeptidation with peptide substrates larger than about 7 residues. If covalent intermediates are involved in the hydrolysis of both large and small substrates, there being no reason to suppose that there is a different mechanism for the large substrates, transpeptidation would be expected. Antonov^® suggested that the lack of (amino) transpeptidation observed with a particular substrate was due to an increase in the

rate of decomposition of the amino enzyme intermediate. It was proposed that the larger substrates give less stable covalent intermediates that are hydrolysed before they can be trapped by a suitable peptide acceptor. Rich^^ proposed that transpeptidation arose due to a slow, structure-dependent release of products from the active site. This idea was further developed by Antonov,^® who calculated a dissociation constant for the enzyme-product complex that is consistent with the rates of transpeptidation seen (see below). Thus, the large substrates may be lost from the active site more rapidly than the smaller substrates or, again, that the covalent intermediates are less stable for large substrates.

More recently transpeptidation studies®® have been used to investigate the incorporation of ^®0-label into substrate and transpeptidation products from H2^®0 . Extensive ""^O-label incorporation into early transpeptidation products challenges the suggestion®® that the incorporation could be due to secondary reactions in the long incubation tim es used by Antonov.®® Thus, these experim ents on transpeptidation provide support for a non-covalent mechanism (see below). The authors also found®® that in H2^®0 only one ^®0 atom was incorporated into the oxygens of the carboxyl group of the C-terminal cleavage product, which implies that the amide cleavage reaction is irreversible.

Other studies on transpeptidation by porcine pepsin have been undertaken by Hofmann et a/.®® Only acyl transpeptidation products were observed and then only after a lag of some minutes, possibly due to a requirement for the dipeptide substrates to bind productively before transpeptidation and release could occur. It was pointed out by Silver and James®"* that a lag in formation of transpeptidation products is incompatible with the formation of an acyl-enzyme intermediate that is subsequently trapped by a suitable acceptor formed during catalysis. This is because if the acyl enzyme intermediate was formed the transpeptidation products should have begun to appear immediately, with no lag.

The only piece of unequivocal evidence in favour of acyl intermediates in aspartic protease catalysis comes from the work of Kaiser®^ on sulfite ester hydrolysis. Here, the acyl intermediate could be trapped with hydroxy lamine. It was shown that the same active site is used for sulfite and peptide hydrolysis, but that only one of the active site aspartates was necessary for sulfitease activity.®^^ The mechanistic

relevance of the observation of sulfitease activity to the physiological reactions of the aspartic proteases remains unclear.