1995
DOI: 10.1021/bi00036a027
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Abstract: Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-91. A 2.5-fold increase in kc,&, was observed on tri-or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in theP2 or P1' position. This preference create… Show more

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Cited by 42 publications
(21 citation statements)
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References 30 publications
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“…The Met residues in the above sequences form the amino terminus of the wild-type protein and are duplicated in the oligonucleotides to maintain the correct reading frame. The PFGRSA sequence was engineered into an internal region in L. donovani ODC surrounding arginine 210 which has previously been shown to be accessible to trypsin (8). Site-directed mutagenesis was performed as described by Kunkel (9).…”
Section: Methodsmentioning
confidence: 99%
“…The sequence PFGRSA was placed into L. donovani ODC at an internal site at which the wild-type ODC was known to be readily cleaved by a catalytically impaired variant of trypsin, H57A trypsin (8). H57A trypsin hydrolyzes peptide substrates 100-1000-fold less efficiently than does trypsin, which is similar to the 1000-fold difference between t-PA and trypsin.…”
mentioning
confidence: 99%
“…2A), which determine (in)sensitivity to PIs. Experiments on synthetic polypeptide enzymes or 'pepzymes' [29] have provided initial clues on the alteration of the activity and/or specificity resulting from modifying key amino acids [30][31][32][33][34][35][36][37]. It does appear that we cannot yet predict functional properties based on structural features.…”
Section: Molecular Diversity Versus Functional Diversitymentioning
confidence: 99%
“…The power of an enzyme to immobilize substrates is also important in bringing reactants into proximity in multisubstrate reactions (17). Category (25) holds that the orientations of the molecular orbitals of the chemically active groups of the enzyme and substrate are both fixed in position and electronically tuned by the active site; however, the observation that precise orientation of the prereaction bonds is probably unnecessary (26,27) is supported by recent experiments indicating that the roles of active site residues may be taken on by substrate moieties (28)(29)(30)(31)(32) or even by freely diffusing small molecules in solution (33), which are unlikely to be held in the precise positions occupied by the original groups. This evidence tends to shift the emphasis back toward the entropic effects and the more diffuse polar and hydrophobic influences of the binding pocket, as well as such concepts as "induced fit" (34,35) and other transduction phenomena.…”
mentioning
confidence: 99%
“…For example, 34 of 36 peptides assayed with trypsin possessed K m values of <1 mM, with an average value for all substrates of 0.18 mM (9)(10)(11)(23)(24)(25). By contrast, 38 of 39 substrates were cleaved by t-PA with a K m of >1 mM and an average value of 7.8 mM (9,(25)(26)(27), 8 of 11 substrates were cleaved by u-PA with a K m of >1 mM and an average value of 2.6 mM (26,27), and 8 of 13 substrates were cleaved by plasmin with a K m of >1 mM and an average value of 1.7 mM (11).…”
Section: Staphylococcal Nuclease As a Model Protease Substratementioning
confidence: 99%