Structure-Function Relationships of Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes

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It is well known that carboxyl proteinases are commonly inhibited by pepstatin^<1)>, DAN^<2)> and EPNP^<3)>, and their catalytic residues are composed of two aspartic acid residues. Thus, carboxyl proteinases are termed aspartic proteinases. These enzymes are highly homologous in both the primary and tertiary structures. On the contrary, we have isolated novel carboxyl proteinases from fungi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP.These enzymes were tentatively named pepstatin- insensitve carboxyl proteinases.In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryote cells. We foucussed our studies on carboxyl proteinases from Pseudomonas sp.101 (PCP) and Xanthomonas sp.T-22 (XCP). PCP and XCP are the first and second carboxyl proteinases isolated from prokaryote cells. The primary structures of PCP and XCP does not have any homologous sturucture to those of aspartic … More proteinases (pepstatin-insensitive carboxyl proteinase) reported so far. Moreover, the well-conserved structure, -Asp**-Thr-Gly- (Asp** : catalytic residue) in the active center of aspartic proteinases was not observed.The following results were obtained.1.Identification of Catalytic Residues by Using Site-directed Mutagenesis TechniquePCP (372 amino acid residues) and XCP (398 amino acid residues) have 52% homology to each other. Based on the high sequence homology, eight amino acid residues for catalytic residues (aspartic or gultamic residues) were piked up, and all of them were mutated to alanine residues. We analyzed these alanine mutants for both auto-catalytic processing ability and proteinase activity. Consequently, D170, E217, E222, and D328 (PCP numbering) are strongly suggested to be the candidates for the catalytic residues. Probably, a pair of them, which are closely related in tertiary structure constitutes the catalytic residues.2.Identification of Catalytic Residues by Using Tyrostatin DerivativesIn our attempt to use inhibitor in the study of active center, we had isolated a novel inhibitor, tyrostatin (N-isovaleryl-tyrosyl-leucyl-tyrosinal, Ki=2.5 nM for PCP and XCP) from kitasatosporia sp.No.55. Based on the chemical structure, we succeeded in synthesizing a compeptive inhibitor, available for probing the catalytic residues of PCP (N-benzyloxycarbonyl-L-phenylalanine-2,3-epoxypropyl ester).Accordingly, we are in a position to identify the catalytic residues at both of DNA and protein levels. We hope that we will be able to identify the catalytic residues of PCP and XCP in 1996.1)pepstatin, pepsin inhibitor ; 2) DAN,diazoacetyl-DL-norleucine methylester ; 3) EPNP,1,2-epoxy-3-(p-nitrophenoxy) propane. Less