Identification of functional conserved residues of CTP:glycerol-3-phosphate cytidylyltransferase. Role of histidines in the conserved HXGH in catalysis.
J Biol Chem
; 272(24): 15161-6, 1997 Jun 13.
Article
em En
| MEDLINE
| ID: mdl-9182537
ABSTRACT
The CTPglycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTPphosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The kcat values for H14A and H17A were decreased by factors of 5 x 10(-5) and 4 x 10(-4), respectively, with no significant change in Km values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyl-tRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 x 10(-3). Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with kcat values decreasing by factors of 0.14-4 x 10(-4). The Km values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6-130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases, i.e. to stabilize a pentacoordinate transition state.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sequência Conservada
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Histidina
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Nucleotidiltransferases
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA