The Role of Tyrosine 207 in the Reaction Catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Cherie Andrade,Ana M Jabalquinto,Emilio Cardemil,Carolina Sepulveda

doi:10.4067/s0716-97602010000200007

Cherie Andrade, Ana M Jabalquinto + Show 2 more

Open Access

https://doi.org/10.4067/s0716-97602010000200007

Copy DOI

Journal: Biological research	Publication Date: Jan 1, 2010
Citations: 3	License type: cc-by

Affiliation: University of Santiago Chile

Abstract

The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO₂, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO₂ and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.

Highlights

Enzymes have evolved different strategies to catalyze carboxylation and decarboxylation reactions, some enzymes use organic or inorganic cofactors, and others are cofactor independent (Liu & Zhang, 2005)
Two classes of PEP carboxykinases exist in nature: ATP-dependent enzymes, which are found in plants, yeast, trypanosomatids, and some bacteria (Matte et al, 1997), and GTP-dependent PEP carboxykinases found in animals and some bacteria (Hanson & Patel, 1994; Fukuda et al, 2004)
To better understand the importance of the invariant Tyr207 of S. cerevisiae PEP carboxykinase, this residue was changed to leucine, so as to entirely eliminate the ability to form an anion-quadrupole interaction, or an H- bond

Summary

Introduction

Enzymes have evolved different strategies to catalyze carboxylation and decarboxylation reactions, some enzymes use organic or inorganic cofactors, and others are cofactor independent (Liu & Zhang, 2005). Structural (Dunten et al, 2002) and kinetic studies (Dharmarajan et al, 2008) indicate that the equivalent Tyr 235 of human cytosolic PEP carboxykinase establishes an anion-quadrupole interaction with PEP carboxylate. We use site directed mutagenesis to evaluate the contribution of Tyr 207 to substrate affinity and catalysis of Saccharomyces cerevisiae PEP carboxykinase.

Results

Conclusion