X-ray crystallographic study of the binding of peptide chloromethyl ketone inhibitors to subtilisin BPN

Robertus, J.D.; Alden, Richard A.; Birktoft, Jens J.; Kraut, Joseph; Powers, James C.; Wilcox, Philip E.

doi:10.1021/bi00763a009

Cited by 180 publications

(109 citation statements)

References 26 publications

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“…The catalytic efficiency is only slightly decreased when the zinc metallopeptidases neprilysin and thermolysin are considered (Tables 1, 2, 4)., but it is abolished by 2 3 orders of magnitude when the serine proteases c~-chymotrypsin and subtilisin Carlsberg are employed (Tables 3 and 4). Early X-ray crystallographic studies of ~-chymotrypsin [24] and subtilisin BPN' [25] with irreversible chloromethylketone extended peptide inhibitors suggest that extended peptide substrates bind to the active site of these enzymes as in an antiparallel fl-pleated sheet. In this proposal, two hydrogen bonds are contributed by the backbone NH and CO groups of the substrate residue at position P3 and one hydrogen bond is contributed by the backbone NH of the substrate residue at position Pl [24,25].…”

Section: Resultsmentioning

confidence: 99%

“…Early X-ray crystallographic studies of ~-chymotrypsin [24] and subtilisin BPN' [25] with irreversible chloromethylketone extended peptide inhibitors suggest that extended peptide substrates bind to the active site of these enzymes as in an antiparallel fl-pleated sheet. In this proposal, two hydrogen bonds are contributed by the backbone NH and CO groups of the substrate residue at position P3 and one hydrogen bond is contributed by the backbone NH of the substrate residue at position Pl [24,25]. No hydrogen bonding with the enzyme is assigned to the substrate residue at P2.…”

Section: Resultsmentioning

confidence: 99%

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Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

et al. 1996

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Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyMeucine 4-nitroanilide or 3-carboxypropanoylalanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (kcat > 102 s-Z; kcatlKm >-106 s -~ "M-~) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (kcat ~ I s-~-34 s-~). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and thermolysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

et al. 1996

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“…Second, binding energy and specificity are dominated by SI in chymotrypsins (Perona & Craik, 1995), but in subtilisins SI and S4 interactions contribute almost equally (Morihara et al, 1970;Robertus et al, 1972;G r m & Breddam, 1992;G r~n et al, 1992). We chose to examine SCARL and PROK because they are the most distantly related members of the subtilisin family whose structures are known (44% sequence identity, 0.888 A RMS deviation of superimposed Ca-atoms [Siezen et al, 19911).…”

Section: Discussionmentioning

confidence: 99%

Interaction of subtilisins with serpins

et al. 1996

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Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that a'proteinase inhibitor inhibits subtilisin Curlsberg and proteinase K, and alantichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 "C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.

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“…S], which is located in a dead-end pocket in the enzyme (14,16,17). The active sites of the serine proteases are similarly located in clefts (15,18), and in analogy with carboxypeptidase A we might expect dead-end structures in the serine carboxypeptidases. In spite of such conceivable differences between serine proteases and serine carboxypeptidases they share the capability to release alcohols from peptide esters and ammonia and/or the C-terminal amino acid amide from peptide amides (5) because both groups of enzymes possess the required binding sites for these activities: S] in the C-terminal direction, and S~, $2 ..... S. in the N-terminal direction of the scissile bond.…”

Section: Discussionmentioning

confidence: 99%

Influence of guanidine derivatives on the specificity of malt carboxypeptidase

Breddam

Ottesen

1983

Carlsberg Res. Commun.

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Malt carboxypeptidase (Carlsberg Res. Commun. 48, 217-230, 1983) catalyzes hydrolysis of ester substrates with the general formula Bz-X-OMe with preference for substrates where X = Phe, Arg or Lys over those where the X-position is occupied by an amino acid residue with a neutral or acid aliphatic side chain. The rapid hydrolysis of Bz-Phe-OMe is mainly due to a high k~, value while for Bz-Arg-OMe it is mainly due to a low Km value. Addition of salts result in decreased rates of hydrolysis of substrates where X = Lys or Arg (positively charged) and increased rates of hydrolysis of substrates where X = Phe or Leu (hydrophobic).Guanido compounds influence the enzymatic properties of malt carboxypeptidase. Kinetic data for the hydrolysis of various substrates indicate that phenylguanidine binds to the St' binding site of the enzyme in such a manner that the rate of cleavage of ester or amide substrates with large groups in the P( position, i.e. -OEt, -OPt, -OBu, -Ala-OH, -Gly-NH2, is reduced while substrates with small groups in this position i.e. -OMe, -NH2 are hydrolyzed with increased rates. These observations have applications in deamidation reactions since the presence of phenylguanidine in the reaction medium results in a significantly increased yield of the deamidated product.Abbreviations: Ac = acetyl; BiGu = 2-guanidobenzimidazol; Bu = butyl; Bz = benzoyl; DFP = diisopropylphosphorofluoridate; EDTA = ethylene diamine tetraacetic acid sodium salt; FA = furylacryloyl; Gu = guanidine hydrochloride; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; HPLC = high performance liquid chromatography; MeGu = methyl guanidine hydrochloride; Mes = 2-(N-morpholino)ethane sulfonic acid; PhGu = phenylguanidine hydrogencarbonate; PpGu = l-(3-phenylpropylamino) guanidine hydrochloride; Pr = propyl; TEAP = triethyl ammonium phosphate; "Iris = tris(hydroxy methyl) aminomethane; Z = carbobenzoxy. Abbreviations of amino acids, amino acid derivatives and peptides are according to the guidelines of the IUPAC-IUB Commision on Biochemical Nomenclature. The binding notation for the enzyme and the substrates is that of SCHECHTER and BERGER. Accordingly, amino acid residues in the substrate are referred to as P,,P2....P~ in the aminoterminal direction away from the scissible bond and P~ for the carboxy-terminal residue that is hydrolyzed off.

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X-ray crystallographic study of the binding of peptide chloromethyl ketone inhibitors to subtilisin BPN

Cited by 180 publications

References 26 publications

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

Interaction of subtilisins with serpins

Influence of guanidine derivatives on the specificity of malt carboxypeptidase

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