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3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase.
Biochemistry. 2004 May 25; 43(20):5976-86.B

Abstract

The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may attenuate trans-aconitate inhibition of aconitase and other enzymes of the cycle. Although trans-aconitate is a minor endogenous substrate of the Tmt1 enzyme in extracts of S. cerevisiae, the major endogenous substrate has yet to be identified. We show here that a trimethylsilylated derivative of the major methylated endogenous product of Tmt1 in yeast extracts has an identical gas chromatography retention time and an identical electron impact mass spectrum as one of the two possible monomethyl ester derivatives of (2R,3S)-3-isopropylmalate. (2R,3S)-3-Isopropylmalate is an intermediate of the leucine biosynthetic pathway that shares similar intermediates and reaction chemistry with the portion of the citric acid cycle from oxaloacetate to alpha-ketoglutarate via cis-aconitate. The Tmt1 methyltransferase recognizes (2R,3S)-3-isopropylmalate with similar kinetics as it does trans-aconitate, with respective K(m) values of 127 and 53 microM and V(max) values of 59 and 70 nmol min(-1) mg(-1) of protein in a Tmt1-overexpressed yeast extract. However, we found that isopropylfumarate, the direct homologue of trans-aconitate in the leucine biosynthetic pathway, was at best a very poor substrate for the Tmt1 yeast enzyme. Similarly, the direct homologue of 3-isopropylmalate in the citric acid cycle, isocitrate, is also a very poor substrate. This apparent change in specificity between the intermediates of these two pathways can be understood in terms of the binding of these substrates to the active site. These results suggest that the Tmt1 methyltransferase may work in two different pathways in two different ways: for detoxification in the citric acid cycle and for a possibly novel biosynthetic branch reaction of the leucine biosynthetic pathway.

Authors+Show Affiliations

Katz JE
Department of Chemistry and Biochemistry, the Molecular Biology Institute, Los Angeles, California 90095-1570, USA.
Dumlao DS
No affiliation info available
Wasserman JI
No affiliation info available
Lansdown MG
No affiliation info available
Jung ME
No affiliation info available
Faull KF
No affiliation info available
Clarke S
No affiliation info available

MeSH

Aconitic AcidBinding SitesCarboxylic AcidsCitric Acid CycleIsomerismLeucineMalatesMethylationMethyltransferasesMolecular StructureSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsSubstrate Specificity

Pub Type(s)

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

Language

eng

PubMed ID

15147181

Citation

Katz, Jonathan E., et al. "3-Isopropylmalate Is the Major Endogenous Substrate of the Saccharomyces Cerevisiae Trans-aconitate Methyltransferase." Biochemistry, vol. 43, no. 20, 2004, pp. 5976-86.
Katz JE, Dumlao DS, Wasserman JI, et al. 3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase. Biochemistry. 2004;43(20):5976-86.
Katz, J. E., Dumlao, D. S., Wasserman, J. I., Lansdown, M. G., Jung, M. E., Faull, K. F., & Clarke, S. (2004). 3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase. Biochemistry, 43(20), 5976-86.
Katz JE, et al. 3-Isopropylmalate Is the Major Endogenous Substrate of the Saccharomyces Cerevisiae Trans-aconitate Methyltransferase. Biochemistry. 2004 May 25;43(20):5976-86. PubMed PMID: 15147181.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase. AU - Katz,Jonathan E, AU - Dumlao,Darren S, AU - Wasserman,Jacob I, AU - Lansdown,Michael G, AU - Jung,Michael E, AU - Faull,Kym F, AU - Clarke,Steven, PY - 2004/5/19/pubmed PY - 2004/8/24/medline PY - 2004/5/19/entrez SP - 5976 EP - 86 JF - Biochemistry JO - Biochemistry VL - 43 IS - 20 N2 - The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may attenuate trans-aconitate inhibition of aconitase and other enzymes of the cycle. Although trans-aconitate is a minor endogenous substrate of the Tmt1 enzyme in extracts of S. cerevisiae, the major endogenous substrate has yet to be identified. We show here that a trimethylsilylated derivative of the major methylated endogenous product of Tmt1 in yeast extracts has an identical gas chromatography retention time and an identical electron impact mass spectrum as one of the two possible monomethyl ester derivatives of (2R,3S)-3-isopropylmalate. (2R,3S)-3-Isopropylmalate is an intermediate of the leucine biosynthetic pathway that shares similar intermediates and reaction chemistry with the portion of the citric acid cycle from oxaloacetate to alpha-ketoglutarate via cis-aconitate. The Tmt1 methyltransferase recognizes (2R,3S)-3-isopropylmalate with similar kinetics as it does trans-aconitate, with respective K(m) values of 127 and 53 microM and V(max) values of 59 and 70 nmol min(-1) mg(-1) of protein in a Tmt1-overexpressed yeast extract. However, we found that isopropylfumarate, the direct homologue of trans-aconitate in the leucine biosynthetic pathway, was at best a very poor substrate for the Tmt1 yeast enzyme. Similarly, the direct homologue of 3-isopropylmalate in the citric acid cycle, isocitrate, is also a very poor substrate. This apparent change in specificity between the intermediates of these two pathways can be understood in terms of the binding of these substrates to the active site. These results suggest that the Tmt1 methyltransferase may work in two different pathways in two different ways: for detoxification in the citric acid cycle and for a possibly novel biosynthetic branch reaction of the leucine biosynthetic pathway. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15147181/3_Isopropylmalate_is_the_major_endogenous_substrate_of_the_Saccharomyces_cerevisiae_trans_aconitate_methyltransferase_ DB - PRIME DP - Unbound Medicine ER -