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Titolo:
Mechanism of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor: Properties of the P1G, P1A, Y95F, and N97A mutants
Autore:
Stamps, SL; Taylor, AB; Wang, SC; Hackert, ML; Whitman, CP;
Indirizzi:
Univ Texas, Coll Pharm, Dept Chem & Biochem, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 Dept Chem & Biochem, Austin, TX 78712 USA Univ Texas, Coll Pharm, Div Med Chem, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 Pharm, Div Med Chem, Austin, TX 78712 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 32, volume: 39, anno: 2000,
pagine: 9671 - 9678
SICI:
0006-2960(20000815)39:32<9671:MOTPTA>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
AMINO-TERMINAL PROLINE; SITE ARGININE RESIDUES; 4-OXALOCROTONATE TAUTOMERASE; CRYSTAL-STRUCTURES; ANGSTROM RESOLUTION; ENZYMATIC-ACTIVITY; PROTEIN MODELS; FACTOR MIF; REFINEMENT; MUTATIONS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
50
Recensione:
Indirizzi per estratti:
Indirizzo: Whitman, CP Univ Texas, Coll Pharm, Dept Chem & Biochem, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 Biochem, Austin, TX 78712 USA
Citazione:
S.L. Stamps et al., "Mechanism of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor: Properties of the P1G, P1A, Y95F, and N97A mutants", BIOCHEM, 39(32), 2000, pp. 9671-9678

Abstract

Phenylpyruvate tautomerase (PPT) has been studied periodically since its activity was first described over forty years ago. In the last two years, the mechanism of PPT has been investigated more extensively because of the discovery that PPT is the same protein as the immunoregulatory cytokine knownas macrophage migration inhibitory factor (MIF). The mechanism of PPT is likely to involve general base-general acid catalysis. While several lines of evidence implicate Pro-1 as the general base, the identity of the generalacid remains unknown. Crystal structures of MIF with the competitive inhibitor (E)-2-fluoro-p-hydroxycinnamate bound in the active site and that of the protein complexed with the enol form of a substrate, (p-hydroxyphenyl)pyruvate, suggest that Tyr-95 is the only candidate in the vicinity that can function as a general acid catalyst. Although Tyr-95 is nearby the bound inhibitor and substrate, it is not within hydrogen bonding distance of eitherligand. In this study, Tyr-95 was mutated to phenylalanine, and the kinetic and structural properties of the Y95F mutant were determined. This alteration produces a fully active enzyme, which shows no significant structural changes in the active site. The results indicate that Tyr-95 does not function as the general acid catalyst in the reaction catalyzed by wild-type PPT. The mechanism of PPT was studied further by constructing and characterizing the kinetic properties of two mutants of Pro-1 (P1G and P1A) and one mutant of Asn-97 (N97A). The mutation of Asn-97, a residue implicated in the binding of the phenolic hydroxy group of the keto and enol isomers of (p-hydroxyphenyl)pyruvate and of (E)-2-fluoro-p-hydroxycinnamate affects only thebinding affinity of the inhibitor. However, the mutations of Pro-1 have a profound effect on the values of k(cat) and k(cat)/K-m and clearly show that Pro-1 is a critical residue in the reaction. The results are discussed interms of a mechanism in which Pro-1 functions as both the general acid andthe general base catalyst.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 01/10/20 alle ore 16:06:10