Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5 .ANG. resolution

Bott, R.; Subramanian, E.; Davies, David R.

doi:10.1021/bi00269a052

Cited by 193 publications

(101 citation statements)

References 32 publications

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“…Firstly, the replacement of the water or hydroxonium ion capable of hydrogen bonding with the negatively charged aspartates by the carbonyl oxygen of the substrate peptide [24] would be unfavourable. On the other hand, replacement by the hydroxyl of a pepstatin inhibitor which could form a hydrogen bond analogous to that of water would cause no difficulty.…”

Section: Resultsmentioning

confidence: 99%

The active site of aspartic proteinases

1984

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The active site of the aspartic proteinase, endothiapepsin, has been detined by X-ray analysis and restrained least-squares refinement at 2.1 A resolution with a crystallographic agreement value of 0.16. The environments of the two catalytically important aspartyl groups are remarkably similar and the contributions of the NH2-and COOH-terminal domains to the catalytic centre are related by a local 2-fold axis. The carboxylates of the aspartyls share a hydrogen bond and have equivalent contacts to a bound water molecule or hydroxonium ion lying on the local diad. The main chains around 32 and 215 are connected by a novel interaction involving diad-related threonines. It is suggested that the two pK,values of the active site aspartyls arise from a structure not unlike that in maleic acid with a hydrogen-bonded intermediate species and a dicarboxylate characterised by electrostatic repulsions between the two negatively charged groups.

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Section: Resultsmentioning

confidence: 99%

The active site of aspartic proteinases

1984

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“…The assignments for Sd-Si were refined and largely confirmed by direct X-ray studies of inhibitors bound to penicillopepsin [28] and to rhizopuspepsin [27]. There are no direct studies which unequivocally define S{-S;, but the original assignments have been improved by more precise modelling using the refined coordinates of endothiapepsin.…”

Section: Human Renin Human Pepsinmentioning

confidence: 91%

“…The residues P4-Pi were placed by analogy with the inhibitors bound to rhizopuspepsin [27] and penicillopepsin [28]. This brought the scissile bond into close juxtaposition with a water molecule bound to the two active site aspartates [29].…”

Section: Methodsmentioning

confidence: 99%

Computer graphics modelling of human renin

et al. 1984

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A model has been constructed using computer graphics for human renin based on the sequence derived from that of the gene and the 3-dimensional structure defined at high resolution for other homologous aspartic proteinases. Human renin can adopt a 3-dimensional structure close to that of other aspartic proteinases, in which amino acids corresponding to intron-exon junctions in the gene are at surface regions in the 3-dimensional structure. As expected, the essential catalytic residues are retained and the nearby residue 304 is alanine as in the mouse sequence, supporting the idea that Asp 304 of other aspartic proteinases may contribute to the low pH of their optimal activity. There are interesting differences at subsite S; which may contribute to the specificity of human renin. Certain residues at the surface of the enzyme adjacent to the active site cleft are unique to renins and may play a role in recognition and binding of angiotensinogen.

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“…Recent NMR studies, indicating that water; rather than a carboxyl group, is the nucleophile, have provided additional evidence against covalent intermediate formation during catalysis by the aspartic proteases [14]. X-ray crystallographic studies have also suggested that the formation of--a covalent acyl-or amino-enzyme is not feasible because of steric hindrance [8,15].…”

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confidence: 99%

The mechanism of action of aspartic proteases involves ‘push‐pull’ catalysis

Polgár

1987

FEBS Letters

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In accord with the available kinetic and X-ray crystallographic data, it is proposed that the two catalytically competent carboxyl groups of aspartic proteases constitute a functional unit which mediates the proton from the attacking water molecule to the leaving nitrogen atom of the substrate. Protonation of this nitrogen atom has been the main issue of the previous mechanistic proposals. The first step of the present mechanism involves proton transfer from the water to the aspartic diad and concurrently another proton transfer from the diad to the carbonyl oxygen of the scissile peptide bond. These proton transfers provide the driving force for the bond formation between the substrate and water, which leads to the formation of a tetrahedral intermediate. The intermediate breaks down to products by a similar facilitation, i.e. by concerted general acid-base catalysis, which involves simultaneous proton transfers from the intermediate to the diad and from the diad to the leaving nitrogen of the substrate. The symmetrical mechanism of the formation and decomposition of the tetrahedral adduct resembles that found in the serine protease catalysis.Pepsin; Aspartic protease; Enzyme mechanism Aspartic proteases include several important enzymes, such as pepsin, chymosin, renin, cathepsin D and the proteases isolated from numerous fungi. The amino acid sequences of all these enzymes are homologous, in particular around the active-site residues. Comprehensive reviews on aspartic proteases have been published [1][2][3]. The threedimensional structure of pepsin [4] and three microbial aspartic proteases including penicillopepsin [5,6], Rhizopus chinensin protease [7] and Endothiaparasitica protease [7] has been reported. The tertiary structures of pepsin and the microbial Correspondence address: L. Polg~ir,

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Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5 .ANG. resolution

Cited by 193 publications

References 32 publications

The active site of aspartic proteinases

The active site of aspartic proteinases

Computer graphics modelling of human renin

The mechanism of action of aspartic proteases involves ‘push‐pull’ catalysis

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