Rhizopus chinensis acid protease

The Rhizopus chinensis acid protease was obtained from Miles/ Seikagaku. The crystallizing conditions reported by Swan (8) were modified by the addition of 50 mM cacodylate to ensure better buffering action. Large crystals 0.6 x 0.5 x 0.4 mm were obtained routinely by the use of ultra-filtration before the solutions were placed in the refrigerator for crystallization. The crystals are orthorhombic, with a = 60.3, b = 60.7 and c = 107.0 A and one... 

R. Delaney, University of Oklahoma Health Sciences Center, Oklahoma City, Three Molecular Forms of Rhizopus chinensis Acid Protease . 

The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

Matthews et al. have determined the structure of the extracellular proteolytic enzyme, thermolysin, to 2.3 A resolution. This enzyme of molecular weight 37 500 contains one zinc and four calcium ions, and is interesting because of its imusual heat stability. The active site contains a zinc atom tetrahedrally co-ordinated to His-142, His-146, Glu-166, and a water molecule, and in this way resembles carboxypeptidase A. The precise details of the co-ordination of the calcium ions are not reported. However, it is of interest that two have a centre-to-centre distance of 3.8 A and are located within an interior region of the protein, surrounded by carbonyl and carboxylate groups. Loss of calcium does not hinder proteolysis at room temperature, but the enzyme is no longer heat stable. Preliminary X-ray data are also reported for the acidic protease from the fungus Rhizopus chinensis. ... 

THE CRYSTAL STRUCTURE OF AN ACID PROTEASE FROM RHIZOPUS CHINENSIS AT 2.5 A RESOLUTION... 

The acid-protease from Rhizopus chinensis was first isolated by Fukumoto, Tsuru, and Yamamoto (1). Its optimum pH for catalytic activity was shown to be between 2.9 and 3.3 (1). Although its complete sequence has not yet been determined, some limited sequence data are available - particularly that of the 39 amino-terminal residues (2,3) and of the residues in the immediate vicinity of the catalytically active aspartic acid residues (4,5). These data show that this enzyme has substantial sequence homology with porcine pepsin. Investigations of the kinetics of catalysis (6,7) have led to proposals of an extended subsite specificity. 

Figure 1 Composite of a few sections from the 2.5 A electron density map of the acid protease from Rhizopus chinensis, which shows four anti-parallel 3-strands. Some of the prominent side chains are labeled. TThe numbers used correspond to the pepsin sequence).

This paper contains a preliminary report of the crystal structure of the acid protease from Rhizopus chinensis at 2.5 A resolution. The molecule is bilobal with a large cleft between the lobes. Pepstatin binds in the cleft near the catalytically active Asp-35. The overall folding of the molecule consists primarily of antiparallel 3-strands, there being only four small helices. 

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