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Kinetic characterization of wild-type and S229A mutant MurB: evidence for the role of Ser 229 as a general acid.
Biochemistry. 1997 Jan 28; 36(4):796-805.B

Abstract

X-ray derived structural data predicted that serine 229 was positioned to act as a proton donor to the developing C2 carbanion during the reduction of enolpyruvyl-UDP-N-acetylglucosamine catalyzed by the bacterial peptidoglycan biosynthetic flavoenzyme MurB. To investigate this effect, a mutant where serine 229 was replaced by alanine was constructed and purified. Kinetic analysis of the two half-reactions for the mutant enzyme revealed a 9-fold decrease in the reduction of EFlox by NADPH and a dramatic 10(7)-fold decrease in the reoxidation of EFlred with the enolpyruvyl substrate. In addition, studies of S229A with the substrate analog, (E)-enolbutyryl-UDP-N-acetylglucosamine, showed a striking bias of the partitioning toward formation of the (Z) geometric isomer as opposed to formation of the reduced product UDP-methylmuramic acid, which was the predominant product in wild-type MurB. These studies provide evidence for the proposed role of this active-site serine as a general acid catalyst.

Authors+Show Affiliations

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9020777

Citation

Benson, T E., et al. "Kinetic Characterization of Wild-type and S229A Mutant MurB: Evidence for the Role of Ser 229 as a General Acid." Biochemistry, vol. 36, no. 4, 1997, pp. 796-805.
Benson TE, Walsh CT, Massey V. Kinetic characterization of wild-type and S229A mutant MurB: evidence for the role of Ser 229 as a general acid. Biochemistry. 1997;36(4):796-805.
Benson, T. E., Walsh, C. T., & Massey, V. (1997). Kinetic characterization of wild-type and S229A mutant MurB: evidence for the role of Ser 229 as a general acid. Biochemistry, 36(4), 796-805.
Benson TE, Walsh CT, Massey V. Kinetic Characterization of Wild-type and S229A Mutant MurB: Evidence for the Role of Ser 229 as a General Acid. Biochemistry. 1997 Jan 28;36(4):796-805. PubMed PMID: 9020777.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic characterization of wild-type and S229A mutant MurB: evidence for the role of Ser 229 as a general acid. AU - Benson,T E, AU - Walsh,C T, AU - Massey,V, PY - 1997/1/28/pubmed PY - 1997/1/28/medline PY - 1997/1/28/entrez SP - 796 EP - 805 JF - Biochemistry JO - Biochemistry VL - 36 IS - 4 N2 - X-ray derived structural data predicted that serine 229 was positioned to act as a proton donor to the developing C2 carbanion during the reduction of enolpyruvyl-UDP-N-acetylglucosamine catalyzed by the bacterial peptidoglycan biosynthetic flavoenzyme MurB. To investigate this effect, a mutant where serine 229 was replaced by alanine was constructed and purified. Kinetic analysis of the two half-reactions for the mutant enzyme revealed a 9-fold decrease in the reduction of EFlox by NADPH and a dramatic 10(7)-fold decrease in the reoxidation of EFlred with the enolpyruvyl substrate. In addition, studies of S229A with the substrate analog, (E)-enolbutyryl-UDP-N-acetylglucosamine, showed a striking bias of the partitioning toward formation of the (Z) geometric isomer as opposed to formation of the reduced product UDP-methylmuramic acid, which was the predominant product in wild-type MurB. These studies provide evidence for the proposed role of this active-site serine as a general acid catalyst. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9020777/Kinetic_characterization_of_wild_type_and_S229A_mutant_MurB:_evidence_for_the_role_of_Ser_229_as_a_general_acid_ L2 - https://doi.org/10.1021/bi962220o DB - PRIME DP - Unbound Medicine ER -