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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 isomerization 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. All 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. [ABSTRACT FROM AUTHOR]

The observation of binding synergism has been successfully extended to include carboxypeptidases A and B. The behaviour of these two enzymes follows the same pattern previously found for three other Zn-proteases. Thus in all cases examined, the affinity of a suitable Zn-ligand is increased in the presence of a compound (specificity probe) which contains the key structural features of specific substrates. A bifunctional ligand such as phosphonoacetate is particularly useful for generating synergism in both carboxypeptidases. Presumably the carboxylate moiety binds to the C-terminal recognition site while the other functional group interacts with the metal ion. Several basic compounds (e.g. methyl guanidine) act as effective specificity probes for carboxypeptidase B while phenol and other hydrophobic substances serve this purpose in carboxypeptidase A. The above phenomenon appears to be a mechanism designed to enhance catalytic efficiency through a substrate-induced conformational change. We postulate that there is a requirement for at least one ionizable group at the active site. The proposed mechanism keeps this group in the correct ionization state in the presence of water and increases its reactivity alter exclusion of water by substrate binding. We suggest the term xerophilic shift for this process. Since proton transfer is a common process in enzyme reactions, the xerophilic-shift mechanism may play a similar role in many instances. It should therefore be possibe to detect binding synergism in a wide variety of enzymes. [ABSTRACT FROM AUTHOR]

Individual subunits of yeast transaldolase were studied by the method of matrix-bound derivatives. Native dimeric transaldolase was bound to Sepharose activated with low amounts of cyanogen bromide to give matrix-bound transaldolase in which the majority of molecules were bound covalently via only one subunit. Exhaustive washing with 6 M guanidinium chloride and renaturation in triethanolamine-EDTA buffer resulted in a derivative (bound-subunit transaldolase) which retained 65% of the original protein and 63% of the original activity. This active form of transaldolase is more sensitive to 2 M urea than bound-dimeric transaldolase. Bound-subunit transaldolase is able to associate with "nascent" soluble transaldolase subunits generated in situ by diluting a small aliquot of guanidinium-chloride-denatured transaldolase into a much larger volume of buffer. The ability of bound-subunit derivative to pick up soluble "nascent" transaldolase subunits is highly specific and reaches a plateau level close to the original protein and activity content of bound transaldolase. The product of such treatment (bound-renatured transaldolase) has properties virtually indistinguishable from those of bound transaldolase. These results indicate that individual subunits of transaldolase are catalytically active. Bound-subunit transaldolase has Km values similar to those of the bound-dimeric derivatives and can be inactivated with sodium borohydride in the presence of the substrate fructose 6-phosphate. However, the pH profile of the subunit form is substantially altered suggesting a shift in the pKa of an essential protonated group from 9.2 to 8.6. [ABSTRACT FROM AUTHOR]