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| | {{STRUCTURE_1spa| PDB=1spa | SCENE= }} | | {{STRUCTURE_1spa| PDB=1spa | SCENE= }} |
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| - | '''ROLE OF ASP222 IN THE CATALYTIC MECHANISM OF ESCHERICHIA COLI ASPARTATE AMINOTRANSFERASE: THE AMINO ACID RESIDUE WHICH ENHANCES THE FUNCTION OF THE ENZYME-BOUND COENZYME PYRIDOXAL 5'-PHOSPHATE'''
| + | ===ROLE OF ASP222 IN THE CATALYTIC MECHANISM OF ESCHERICHIA COLI ASPARTATE AMINOTRANSFERASE: THE AMINO ACID RESIDUE WHICH ENHANCES THE FUNCTION OF THE ENZYME-BOUND COENZYME PYRIDOXAL 5'-PHOSPHATE=== |
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| - | ==Overview==
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| - | Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_1610831}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 1610831 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_1610831}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Miyahara, I.]] | | [[Category: Miyahara, I.]] |
| | [[Category: Yano, T.]] | | [[Category: Yano, T.]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 08:58:56 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 03:52:31 2008'' |
Revision as of 00:52, 29 July 2008
Template:STRUCTURE 1spa
ROLE OF ASP222 IN THE CATALYTIC MECHANISM OF ESCHERICHIA COLI ASPARTATE AMINOTRANSFERASE: THE AMINO ACID RESIDUE WHICH ENHANCES THE FUNCTION OF THE ENZYME-BOUND COENZYME PYRIDOXAL 5'-PHOSPHATE
Template:ABSTRACT PUBMED 1610831
About this Structure
1SPA is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate., Yano T, Kuramitsu S, Tanase S, Morino Y, Kagamiyama H, Biochemistry. 1992 Jun 30;31(25):5878-87. PMID:1610831
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