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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
2
|
| pubmed:dateCreated |
1997-9-8
|
| pubmed:abstractText |
L-Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active-site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recombinant human L-arginine:glycine amidinotransferase were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28-nm, 0.29-nm and 0.236-nm resolution, respectively. The mutation of active-site residues which are involved in substrate-binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000-fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild-type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition-state stabilization, and not due to major conformational rearrangements of the protein. These results show that a His-Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain-like cysteine proteinases.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Jul
|
| pubmed:issn |
0014-2956
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
15
|
| pubmed:volume |
247
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
483-90
|
| pubmed:dateRevised |
2007-7-23
|
| pubmed:meshHeading |
pubmed-meshheading:9266688-Amidinotransferases,
pubmed-meshheading:9266688-Amino Acid Sequence,
pubmed-meshheading:9266688-Binding Sites,
pubmed-meshheading:9266688-Catalysis,
pubmed-meshheading:9266688-Cloning, Molecular,
pubmed-meshheading:9266688-Crystallography, X-Ray,
pubmed-meshheading:9266688-DNA,
pubmed-meshheading:9266688-Humans,
pubmed-meshheading:9266688-Kinetics,
pubmed-meshheading:9266688-Models, Molecular,
pubmed-meshheading:9266688-Models, Structural,
pubmed-meshheading:9266688-Mutagenesis, Site-Directed,
pubmed-meshheading:9266688-Point Mutation,
pubmed-meshheading:9266688-Protein Conformation,
pubmed-meshheading:9266688-Recombinant Proteins
|
| pubmed:year |
1997
|
| pubmed:articleTitle |
Substrate binding and catalysis by L-arginine:glycine amidinotransferase--a mutagenesis and crystallographic study.
|
| pubmed:affiliation |
Max-Planck-Institut für Biochemie, Martinsried, Germany.
|
| pubmed:publicationType |
Journal Article
|