<i>Rhizopus</i> Acid Proteinases (Rhizopus-pepsins): Properties and Homology with Other Acid Proteinases

Graham, Janease Erin; Sodek, Jaro; Hofmann, Theo

doi:10.1139/o73-098

Cited by 31 publications

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“…Recent data on the amino-acid sequences of Rhizopus pepsin (18) and of chymosin (19) show considerable homology to that of pepsin (20,21). These data may be expected to facilitate the xray crystallographic study of the three-dimensional structures of the acid proteinases.…”

Section: Biochemistry: Sampath-kumar and Frutonmentioning

confidence: 85%

Studies on the Extended Active Sites of Acid Proteinases

Sampathkumar

Fruton

1974

Proc. Natl. Acad. Sci. U.S.A.

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The kinetics of the hydrolysis of a series of peptide substrates at a single peptide bond (between L-phenylalanyl and L-phenylalanyl) by the acid. proteinases gastric pepsin A (EC 3.4.23.1), Rhizopus pepsin (EC 3.4.23.9), and beef-spleen cathepsin D ' (EC 3.4.23.5) have been determined by use of the fluorescamine assay method. The results indicate that the extended active site of pepsin can accommodate a sequence of at least seven amino-acid residues. Although the other two acid proteinases appear to act at the sensitive L-phenylalanyl-L-phenylalanyl bond by a mechanism similar to that of pepsin, the influence of structural changes on either side of the sensitive dipeptidyl unit on the kinetic parameters is different from that for pepsin. These data give further evidence for the importance of secondary interactions in determining the catalytic efficiency of enzymes that act on oligomeric substrates.Extensive evidence is now available to indicate that catalysis by some proteinases involves multiple cooperative interaction of an extended region of the enzyme with several amino-acid residues of the oligopeptide substrate, in addition to those directly linked by the sensitive peptide bond (1). The extended active-site region of such a proteinase thus includes not only the enzymic groups concerned with the bond-breaking step in the over-all catalytic process, but also other structural units, at a distance from these catalytic groups, whose "secondary" interactions with the oligopeptide substrate can influence greatly the rate of catalytic action (2, 3).Clearly, the specificity of proteinases that exhibit pronounced effects of secondary enzyme-substrate interaction on their catalytic efficiency cannot be described solely in terms of the amino-acid residues of the substrate at the sensitive bond, as in the preferential action of pancreatic trypsin at amide or ester bonds involving the carbonyl group of ilysyl and iarginyl residues. Instead, additional information must be provided about the effect of structural modification of oligopeptide substrates at positions one or more aminoacid residues on either side of the dipeptidyl unit whose peptide bond is preferentially cleaved by the enzyme.The accumulation of such data on the effect of substrate modification on the kinetics of enzyme action has encouraged efforts to "map" the extended active site of some proteinases (4, 5) by denoting regions of the active site as "subsites," each corresponding to an amino-acid unit of the substrate. Such mapping involves the assumption, however, that the extended active site has little conformational flexibility and that the structural units of the substrate "fit" into a relatively rigid cleft of the kind made evident for lysozyme by x-ray crystallography (6). Although it has been established that several enzymes are catalytically' active in the crystalline state (7), the question of the extent of conformational flexibility at the active sites of dissolved enzymes is open. The possibility exists, therefore, that some proteinas...

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Section: Biochemistry: Sampath-kumar and Frutonmentioning

confidence: 85%

Studies on the Extended Active Sites of Acid Proteinases

Sampathkumar

Fruton

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…In several reports an acidic Rhizopus protease (Rhizopus pepsin; EC 3.4.23.21) has well been characterized [29,30,31,8,9,27,7]. In our program to select strains with an optimized enzyme pattern from Tempeh production Rehms and Barz [21] had screened 46 Rhizopus isolates regarding protease-, a-galactosidase-and phytase activities using different solid selection media.…”

Section: Introductionmentioning

confidence: 99%

Expression of proteases by Rhizopus species during Tempeh fermentation of soybeans

Heskamp

Barz²

1998

Nahrung

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Expression of proteolytic activities by nine Rhizopus strains during soybean fermentation to produce Tempeh has been determined. All strains showed activity at acidic, neutral, and alkaline pH values. In protein solubilizates prepared from Tempeh pronounced differences in the time course and intensity of maximum protease activities were observed between the isolates when measured at pH 7, the approximate pH of Tempeh during ripening. Proteases expressed by fungi during growth on Tempeh were solubilized and the enzymes were enriched by acetone precipitation and chromatographies with Phenylsuperose, Mono S, and Mono P, respectively. They were identified as one aspartic‐ (∼35 kD) and one serine (∼33 kD) protease, each existing in different isoforms. Proteases isolated from Tempeh were identical to those expressed during fungal growth in a soyprotein‐raffinosephytate liquid medium. The temperature stability of the Tempeh‐ and the cell wall‐isolated proteases is very low in contrast to the proteases secreted into the medium. Proteases isolated from Tempeh, cell walls and liquid media exhibit differences in the number of alkaline isoforms.

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“…In recent years acid proteinases from fungi have attracted considerable interest among many enzymologists (see reviews by Sodek & Hofmann, 1970;Matsubara & Feder, 1971;Hofmann, 1975). Partial amino acid sequences of two of them, penicillopepsin from Penicillium janthinellum and Rhizopus-pepsin from Rhizopus chinensis (Graham et al, 1973;Hofmann, 1974) indicate that these enzymes are homologous to porcine pepsin and chymosin. An active site peptide of an acid proteinase from Aspergillus awamori (Lobareva et al, 1972) was shown to be identical with a corresponding peptide of penicillopepsin (Kovaleva et al, 1972).…”

mentioning

confidence: 99%

Aspergillus oryzae acid proteinase. Purification and properties, and formation of π-chymotrypsin

Davidson

Gertler

Hofmann

1975

Biochemical Journal

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An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.

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Rhizopus Acid Proteinases (Rhizopus-pepsins): Properties and Homology with Other Acid Proteinases

Cited by 31 publications

References 0 publications

Studies on the Extended Active Sites of Acid Proteinases

Studies on the Extended Active Sites of Acid Proteinases

Expression of proteases by Rhizopus species during Tempeh fermentation of soybeans

Aspergillus oryzae acid proteinase. Purification and properties, and formation of π-chymotrypsin

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