On the size of the active site in proteases. I. Papain

Schechter, Israël; Berger, Arieh

doi:10.1016/s0006-291x(67)80055-x

Cited by 5,096 publications

(3,006 citation statements)

References 7 publications

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“…These substrates are hydrolyzed at the Ala-Phe and Ala-Leu bonds, respectively, conforming with the primary specificity of the enzyme, known to cleave internal peptide bonds of peptides on the amino side of hydrophobic amino acid residues [1,6,13]. The hydrophobic Phe or Leu residues of the substrates interact with the primary specificity site of the enzyme S'~, whereas the two Ala residues interact with the secondary sub-sites St and S 2 (according to the nomenclature of Schechter and Berger; [21]). Even the 4-nitroanilide group possibly interacts with a sub-site, S~, on the surface of the enzyme.…”

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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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%

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

et al. 1996

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“…a part of the physiological substrate histone H3 and human cathepsin L by crystallizing the complex with a catalytic mutant of the enzyme and elucidating its structure [16]. Refinement confirmed the positioning of only three amino acids from the histone H3 [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] peptide. Our crystal packing analysis suggested that there is not enough space to accommodate all 14 peptide residues in the available space in the active site cleft.…”

Section: Abstract: Cathepsin; Cysteine Cathepsin; Substrate Interactionmentioning

confidence: 99%

Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L

Sosnowski

Turk

2016

FEBS Letters

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Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 A revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity.

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“…The conformation of Pre2 begins to differ from thrombin at Glu 14H, where it has an additional helical turn in thrombin (Figs. 3, S2 binding subsites (Schechter & Berger, 1967) (Freer et al, 1970), which is also accompanied by solvent exposure of buried Glu 192. The unfolding of the turn between Ala 190 and Gly 193 and its subsequent displaced reformation at Cys 191-Asp 194 simultaneously forms most of the S1 specificity subsite.…”

Section: Activation Domainmentioning

confidence: 99%

The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin

et al. 1994

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The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of a-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3 . 7~ compared to the bulk structure). This segment has 2 type I1 0-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192.The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having a n acidic residue in the P 3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugenthrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules. Keywords: activation; exosite binding; hirugen-thrombin; PPACK-thrombin; prethrombin2Prothrombin activation to a-thrombin by the prothrombinase complex (Factor Xa-Factor Va-phospholipids-Ca*+) initially proceeds through cleavage at Arg 320 (Arg 15, chymotrypsinogen numbering; Fig. 1) to give rise to the intermediate product meizothrombin (Krishnaswamy et al., 1987;Mann, 1987 Abbreviations: pre2, prethrombin2; Pre2 (with a capital), hirugenpre2 complex; PPACK, D-Phe-Pro-Arg chloromethyl ketone; Throm, or-thrombin; hirugen (Hir), hirudin 53-64; BPTI, bovine pancreatic trypsin inhibitor; DFP, di isopropylfluorophosphate; PEG, polyethyleneglycol; DAPA, dansyl-arginine N-(3-ethyl-1,5 pentanediy1)amide. This is followed by cleavage at Arg 271 to produce a-thrombin, which autolytically cleaves at Arg 284 to give a-thrombin (des Thr 272-Arg 284) (Downing et al., 1975). The stable form of athrombin is thus composed of the original residues from prothrombin Thr 285 to Arg 320, which corresponds to the A chain, and Ile 321 to Glu 579 in the B chain. In contrast, the activation of human prothrombin to a-thrombin by the catalyst Factor Xa-phospholipid-Ca'+ proceeds by cleavage at Arg 271 that leads to the noncovalently associated products, prothrombin fragment 1.2 (Ala 1-Arg 271) and pre2 (Thr 272-Glu 579). Subsequently, the catalytically inactive pre2 is cleaved at Arg 320 to a-thrombin and, ultimately, to the des Thr 272-Arg 284 cleavage product of a-thrombin. Thus, the simplest precursor form 2254

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On the size of the active site in proteases. I. Papain

Cited by 5,096 publications

References 7 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

Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L

The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin

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