Synergistic binding of hydrophobic probes and zinc ligands to thermolysin

Abstract

The strong synergism previously observed in the binding of inhibitors to two Zn-proteases, has also been found for thermolysin. As in earlier cases, the effects are produced by a small Zn-ligand (e.g. a hydroxamate) in the presence of another compound which contains the key structural features of specific substrates (a specificity probe). For thermolysin, the most effective specificity probes are hydrophobic derivatives of amines and amino acids (e.g. carbobenzyloxy-L-alaninol). Even the simple combination of benzyl alcohol and formohydroxamate displays considerable synergism. The above effects are temperature dependent and correlate well with a thermally induced conformational isornerization reported recently for this enzyme.Our results seem to be related to previous observations of substrate synergism in the reverse reaction and to superactivation by chemical modification of this enzyme. A11 these effects are consistent with a change in the environment of the catalytically important zinc atom upon binding of the hydrophobic side chain of the substrate. With the inclusion of thermolysin, binding synergism is now known to occur in an endopeptidase as well as in exopeptidases of diverse specificity. The general occurrence of this phenomenon in zinc proteases and its possible significance are discussed in an accompanying study.Previously we reported the synergistic interactions of ligands with microsomal aminopeptidase (MAP ; DiGregorio et al., 1988) and the angiotensin-converting enzyme (ACE) (Pfuetzner and Chan, 1988) which were both Zn-containing proteases. In these studies, the synergism could be detected by following the concurrent effects of two inhibitors on enzyme activity. By means of a suitable graphical analysis (Yonetani and Theorell, 1964), it was possible to determine quantitatively the degree to which binding of one inhibitor increased the affinity of the other towards the enzyme. For both of the above enzymes, synergistic binding was found only between a Zn-ligand and a compound containing key structural features of specific substrates. This structural requirement for synergism was particularly striking because of the large difference in specificity between MAP (Wachsmuth et al., 1966) and ACE (Soffer, 1976). We therefore felt that the above effects might be of some general significance. To probe the extent of the phenomenon, we have broadened our investigation to include thermolysin which is also a Znprotease (for a review see Matthews, 1988). This enzyme has been chosen because it would allow us to determine whether the structural requirements for synergism previously observed for exopeptidases would also apply to an endopeptidase. Another advantage of studying thermolysin is that the

General occurrence of binding synergism in zinc proteases and its possible significance

“…The results reported in this study, taken with our previous work on three other Zn-proteases (DiGregorio et al, 1988;Pfuetzner and Chan, 1988;Pfuetzner and Chan, 1993) show a striking pattern of behaviour in this class of enzymes. Thus in all cases examined, synergistic binding to the enzyme requires the presence of a Zn-ligand and a probe with structural characteristics of specific substrates.…”

“…In the case of weak inhibitors or compounds with low solubility, the K, values were often determined more conveniently from the Yonetani-Theorell plot. The procedure is described in the preceding paper (Pfuetzner and Chan, 1993).…”

“…For the sake of consistency, a single K, value is used in the data for each table. Further details concerning these kinetic experiments and the analysis of data can be found in the preceding study (Pfuetzner and Chan, 1993).…”

“…In the preceding study (Pfuetzner and Chan, 1993) we described the detection of binding synergism in thermolysin for certain combinations of inhibitors. As we had observed previously in two other Zn-proteases (DiGregorio et al, 1988;Pfuetzner and Chan, 1988), the effects were produced by a Zn-ligand in the presence of a compound with important structural characteristics of specific substrates (specificity probe).…”

Synergistic binding of inhibitors to the protease from HIV type 1

“…To investigate the possible existence of such interactions between subsites we have examined whether two inhibitors binding to different locations could exert effects on each other. In the past few years our laboratory has investigated the phenomenon of synergism in the binding of inhibitors to several zinc proteases [13][14][15] including the clinically important angiotensinconverting enzyme [16]. Although inhibitor synergism has not been reported in aspartate proteases, theoretical considerations suggest that it should be observable in a variety of enzymes [15].…”