Novel Insights into the Biotin Carboxylase Domain Reactions of Pyruvate Carboxylase from Rhizobium etli

Authors

Tonya N. Zeczycki, University of Wisconsin - MadisonFollow
Ann L. Menefee, Marquette University
Abdussalam Adina-Zada, University of Western Australia
Sarawut Jitrapakdee, Mahidol University
Kathy H. Surinya, University of Adelaide
John C. Wallace, University of Adelaide
Paul V. Attwood, University of Western Australia
Martin St. Maurice, Marquette UniversityFollow
W Wallace Cleland, University of Wisconsin - MadisonFollow

Document Type

Article

Language

eng

Format of Original

14 p.

Publication Date

2011

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO3 − deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO3 −, Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in kcat for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO3 − by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO2 and PO4 3−. PO4 3− then acts as the base to deprotonate the tethered biotin at the N1-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO2 to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain

Comments

Accepted version. Biochemistry, Vol. 50 (2011): 9724-9737. DOI. © 2011 ACS Publications. Used with permission

Recommended Citation

Zeczycki, Tonya N.; Menefee, Ann L.; Adina-Zada, Abdussalam; Jitrapakdee, Sarawut; Surinya, Kathy H.; Wallace, John C.; Attwood, Paul V.; Maurice, Martin St.; and Cleland, W Wallace, "Novel Insights into the Biotin Carboxylase Domain Reactions of Pyruvate Carboxylase from Rhizobium etli" (2011). Biological Sciences Faculty Research and Publications. 106.
https://epublications.marquette.edu/bio_fac/106