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Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase.

ABSTRACT: Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.

SUBMITTER: Barrett AJ

PROVIDER: S-EPMC1149619 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundSedolisins are acid proteases that are related to the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both TriPeptidyl Peptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies.ResultsWe studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows one major clade containing non-fungal sequences only and two major as well as two minor clades containing only fungal sequences. One of the major fungal clades contains all known TPPs whereas the other contains characterized endosedolisins. We identified four Cluster Specific Inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried α-helix that induces a conformational change near the binding pocket. We also identified a total of 15 specificity determining positions (SDPs) of which five, identified in two independent analyses, form highly connected SDP sub-networks. Modeling of virtual mutants suggests a key role for the W307A or F307A substitution. The remaining four key SDPs physically interact at the interface of the catalytic domain and the enzyme's prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. One of the two small fungal clades concerns a subfamily that contains 213 sequences, is mostly similar to the major TPP subfamily but differs, interestingly, in position 307, showing mostly isoleucine and threonine.ConclusionsAnalysis confirms there are at least two sedolisin subfamilies in fungi: TPPs and endopeptidases, and suggests a third subfamily with unknown characteristics. Sequence and functional diversification was centered around buried SDP307 and resulted in a conformational change of the pocket. Mutual Information network analysis forms a useful instrument in the corroboration of predicted SDPs.

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Chicken liver Pz-peptidase, a thiol-dependent metallo-endopeptidase.

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