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An X-ray diffraction analysis has been carried out at 2.5-A resolution of the three-dimensional structure of the Rhizopus chinensis carboxyl proteinase complexed with pepstatin. The resulting model of the complex supports the hypothesis [Marciniszyn, J., Hartsuck, J.A., & Tang, J. (1976) J. Biol. Chem. 251, 7088-7094] that statine (3-hydroxy-4-amino-6-methylheptanoic acid) approaches an analogue of the transition state for catalysis. The way in which pepstatin binds to the enzyme can be extended to provide a model of substrate binding and a model of the transition-state complex. This in turn has led to a proposed mechanism of action based on general acid-base catalysis with no covalent intermediates. These predictions are in general agreement with kinetic studies using several carboxyl proteinases, which together with their sequence homology and their common three-dimensional structures suggest that this mechanism can be extrapolated to all carboxyl proteinases.

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SynopsisA tripeptide molecule, L-alanyl-glycyl-glycine, crystallizes in the form of a left-handed helix with (@,$I = -83", 170". A pseudohexagonal packing arrangement and interchain hydrogen-bonded interactions are reminiscent of the model for the structure of poly(g1ycine)-11. Observations of certain intermolecular interactions appear to be relevant to the stereochemical assumptions incorporated in the models proposed for poly(g1ycine)-I1 and related polypeptides.

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The molecular structures of two fungal acid proteases at 3 A resolution have been compared, and found to have similar secondary and tertiary folding Tse enzymes are bilobal and have a pron c cleft between the two loes. This cleft has been identified as the active site region from inhibitor binding studies. The results of the comparison are discussed in terms of homology among the acid proteases in general. The acid proteases form an interesting and well-defined class of enzymes They include the mammalian enzymes pepsin and chymosin (rennin), and a number of fungal enzymes such as those from Rhizopus chinensis, Endothia parasitica, and Penicillium janthinellum (see refs. 1 and 2). These enzymes are all characterized by molecular weights in the region of 35,000, and pH optima for catalytic action in the range of 1.5-5.0. They share the property of being inhibited by.epoxy compounds and by diazonium compounds in the presence of Cu2+ ions (3,4). Their catalytic activities have been studied extensively and shown to involve two aspartic acid residues [Asp 32 and Asp 215 in the pepsin sequence (5)]. They are all inhibited by a microbial oligopeptide, pepstatin, an acid-protease-specific inhibitor obtained from culture filtrates of Streptomyces (6-9). They all possess essentially similar substrate specificity, preferring hydrophobic residues on both sides of the scissile bond. In addition, their catalytic action appears to involve extended subsite interactions with several amino acid side chains of the substrate (1, 2).The complete amino acid sequence has been determined for porcine pepsin (5), and limited sequence information is available for several other acid proteases (10)(11)(12). These data reveal similarity in sequence and suggest there is homology among the various acid proteases. In particular, the residues immediately adjacent to the two catalytically important aspartic acid residues appear to be identical in all the cases that have been