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Titolo:
CONVERSION OF SERINE-114 TO CYSTEINE-114 AND THE ROLE OF THE ACTIVE-SITE NUCLEOPHILE IN ACYL TRANSFER BY MYRISTOYL-ACP THIOESTERASE FROM VIBRIO-HARVEYI
Autore:
LI J; SZITTNER R; DEREWENDA ZS; MEIGHEN EA;
Indirizzi:
MCGILL UNIV,DEPT BIOCHEM,3655 DRUMMOND ST MONTREAL PQ H3G 1Y6 CANADA MCGILL UNIV,DEPT BIOCHEM MONTREAL PQ H3G 1Y6 CANADA UNIV VIRGINIA,DEPT MOL PHYSIOL & BIOL PHYS CHARLOTTESVILLE VA 22908
Titolo Testata:
Biochemistry
fascicolo: 31, volume: 35, anno: 1996,
pagine: 9967 - 9973
SICI:
0006-2960(1996)35:31<9967:COSTCA>2.0.ZU;2-5
Fonte:
ISI
Lingua:
ENG
Soggetto:
FATTY-ACID SYNTHETASE; RAT MAMMARY-GLAND; THIO ESTER HYDROLASE; PHOTOBACTERIUM-PHOSPHOREUM; BACTERIAL BIOLUMINESCENCE; TRIACYLGLYCEROL LIPASE; LUMINESCENT BACTERIA; DIRECTED MUTAGENESIS; PHENOTYPIC SELECTION; NUCLEOTIDE-SEQUENCE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
56
Recensione:
Indirizzi per estratti:
Citazione:
J. Li et al., "CONVERSION OF SERINE-114 TO CYSTEINE-114 AND THE ROLE OF THE ACTIVE-SITE NUCLEOPHILE IN ACYL TRANSFER BY MYRISTOYL-ACP THIOESTERASE FROM VIBRIO-HARVEYI", Biochemistry, 35(31), 1996, pp. 9967-9973

Abstract

The lux-specific myristoyl-ACP thioesterase (LuxD) is responsible fordiverting myristic acid into the luminescent system and can function as an esterase and transferase as well as cleave myristoyl-CoA and other thioesters, The recently elucidated crystal structure of the enzymeshows that it belongs to the alpha/beta hydrolase family and that it contains a typical catalytic triad composed of Asp(211), His(241), andSer(114). What is unusual is that the nucleophilic S-114 is not contained within the esterase consensus motif GXSXG although the stereochemistry of the turn involving S-114 is almost identical to the nucleophilic elbow found in alpha/beta hydrolases. In contrast to mammalian thioesterases, deacylation of LuxD was the rate-limiting step, with the level of acylated enzyme formed on reaction with myristoyl-CoA and the pre-steady-state burst of p-nitrophenol on cleavage of p-nitrophenyl myristate both being 0.7 mol/mol. Cold chase experiments showed that the deacylation rate of LuxD corresponded closely to the turnover rate of the enzyme with ester or thioester substrates, Replacement of S-114 by a cysteine residue generated a mutant (S114C) that was acylated with the same pH dependence as LuxD but had greatly diminished capacity to transfer acyl groups to water or glycerol, The acyl group could be removed from the S114C mutant by neutral hydroxylamine, showing that a cysteine residue had been acylated, Mutation of H-241 creating the double mutant, S114C . H241N, decreased acylation of the cysteine residue, These results provide direct kinetic and chemical evidence that S-114 is the site of acylation linked to H-241 in the charge relay system and have led to the recognition of a more general consensus motif flanking the nucleophilic serine in thioesterases.

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Documento generato il 25/09/20 alle ore 05:46:11