HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 25, 17238-17245, June 23, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/25/17238    most recent M602299200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Xu, W. Articles by Chollet, R. Search for Related Content PubMed PubMed Citation Articles by Xu, W. Articles by Chollet, R. Social Bookmarking                      What's this?

The Importance of the Strictly Conserved, C-terminal Glycine Residue in Phosphoenolpyruvate Carboxylase for Overall Catalysis

MUTAGENESIS AND TRUNCATION OF GLY-961 IN THE SORGHUM C4 LEAF ISOFORM^*

Wenxin Xu¹, Shaheen Ahmed¹, Hideaki Moriyama, and Raymond Chollet^¶2

From the Department of Biochemistry, Department of Chemistry, and the ^¶Plant Science Initiative, University of Nebraska, Lincoln, Nebraska 68588-0664

Phosphoenolpyruvate carboxylase (PEPC) is a "multifaceted," allosteric enzyme involved in C4 acid metabolism in green plants/microalgae and prokaryotes. Before the elucidation of the three-dimensional structures of maize C4 leaf and Escherichia coli PEPC, our truncation analysis of the sorghum C4 homologue revealed important roles for the enzyme's C-terminal -helix and its appended QNTG⁹⁶¹ tetrapeptide in polypeptide stability and overall catalysis, respectively. Collectively, these functional and structural observations implicate the importance of the PEPC C-terminal tetrapeptide for both catalysis and negative allosteric regulation. We have now more finely dissected this element of PEPC structure-function by modification of the absolutely conserved C-terminal glycine of the sorghum C4 isoform by site-specific mutagenesis (G961(A/V/D)) and truncation (C1/C4). Although the C4 polypeptide failed to accumulate in a PEPC^- strain (XH11) of E. coli transformed with the Asp mutant, the other variants were produced at wild-type levels. Although neither of these four mutants displayed an apparent destabilization of the purified PEPC homotetramer, all were compromised catalytically in vivo and in vitro. Functional complementation of XH11 cells under selective growth conditions was restricted progressively by the Ala, C1 and Val, and C4 modifications. Likewise, steady-state kinetic analysis of the purified mutant enzymes revealed corresponding negative trends in k_cat and k_cat/K_0.5 (phosphoenolpyruvate) but not in K_0.5 or the Hill coefficient. Homology modeling of these sorghum C-terminal variants against the structure of the closely related maize C4 isoform predicted perturbations in active-site molecular cavities and/or ion-pairing with essential, invariant Arg-638. These collective observations reveal that even a modest, neutral alteration of the PEPC C-terminal hydrogen atom side chain is detrimental to enzyme function.

Received for publication, March 10, 2006 , and in revised form, April 18, 2006.

^* This research was supported in part by United States National Science Foundation Grant MCB-0130057 (to R. C.) and was published as No. 15,172 in the University of Nebraska Agricultural Research Division journal series. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These researchers contributed equally to this project and, thus, should be considered as joint first-authors.

² To whom correspondence should be addressed: Dept. of Biochemistry, University of Nebraska-Lincoln, N246 Beadle Center, Lincoln, Nebraska 68588-0664. Tel.: 402-472-2936; Fax: 402-472-7842; E-mail: rchollet1{at}unl.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?