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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
37
|
| pubmed:dateCreated |
1991-10-24
|
| pubmed:abstractText |
The existence of an oxyanion hole in cysteine proteases able to stabilize a transition-state complex in a manner analogous to that found with serine proteases has been the object of controversy for many years. In papain, the side chain of Gln19 forms one of the hydrogen-bond donors in the putative oxyanion hole, and its contribution to transition-state stabilization has been evaluated by site-directed mutagenesis. Mutation of Gln19 to Ala caused a decrease in kcat/KM for hydrolysis of CBZ-Phe-Arg-MCA, which is 7700 M-1 s-1 in the mutant enzyme as compared to 464,000 M-1 s-1 in wild-type papain. With a Gln19Ser variant, the activity is even lower, with a kcat/KM value of 760 M-1 s-1. The 60- and 600-fold decreases in kcat/KM correspond to changes in free energy of catalysis of 2.4 and 3.8 kcal/mol for Gln19Ala and Gln19Ser, respectively. In both cases, the decrease in activity is in large part attributable to a decrease in kcat, while KM values are only slightly affected. These results indicate that the oxyanion hole is operational in the papain-catalyzed hydrolysis of CBZ-Phe-Arg-MCA and constitute the first direct evidence of a mechanistic requirement for oxyanion stabilization in the transition state of reactions catalyzed by cysteine proteases. The equilibrium constants Ki for inhibition of the papain mutants by the aldehyde Ac-Phe-Gly-CHO have also been determined. Contrary to the results with the substrate, mutation at position 19 of papain has a very small effect on binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Sep
|
| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
17
|
| pubmed:volume |
30
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
8924-8
|
| pubmed:dateRevised |
2000-12-18
|
| pubmed:meshHeading |
pubmed-meshheading:1892809-Amino Acid Sequence,
pubmed-meshheading:1892809-Anions,
pubmed-meshheading:1892809-Base Sequence,
pubmed-meshheading:1892809-Catalysis,
pubmed-meshheading:1892809-Glutamine,
pubmed-meshheading:1892809-Hydrolysis,
pubmed-meshheading:1892809-Kinetics,
pubmed-meshheading:1892809-Molecular Sequence Data,
pubmed-meshheading:1892809-Oxidation-Reduction,
pubmed-meshheading:1892809-Papain,
pubmed-meshheading:1892809-Protein Conformation,
pubmed-meshheading:1892809-Substrate Specificity
|
| pubmed:year |
1991
|
| pubmed:articleTitle |
Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain.
|
| pubmed:affiliation |
Biotechnology Reserach Institute, National Research Council of Canada, Montréal, Québec.
|
| pubmed:publicationType |
Journal Article
|