Báo cáo khoa học: Critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme

Critical Roles Of Conserved Carboxylic Acid Residues In Pigeon Cytosolic NADP+-Dependent Malic Enzyme

Authors: Shuo-Chin Chang, Kuan-Yu Lin, Yu-Jung Chen, Chin-Hung Lai, Gu-Gang Chang, Wei-Yuan Chou

Field: Biochemistry

Document Content: This study investigates the critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme. Through site-directed mutagenesis and kinetic analysis, researchers aimed to clarify the mechanism of this enzyme, which catalyzes the reduction of NADP+ to NADPH and the decarboxylation of L-malate to pyruvate via a general acid/base mechanism. The research involved mutating four conserved acidic amino acids (E234, D235, D257, and D258) to alanine and analyzing the kinetic properties of the resulting mutant enzymes. Key findings indicate that E234 is crucial for bivalent metal ion binding, while D235 and D258 play significant roles in catalysis. Specifically, D258 is proposed to function as a general base, facilitating the extraction of a proton from the C2 hydroxyl group of L-malate, thereby promoting oxaloacetate formation and hydride transfer. The study also explores the interaction of these residues with metal ions and their contribution to the enzyme’s overall catalytic efficiency.

Detailed Table of Contents:

• Keywords
• Correspondence
• Abstract
• Introduction
• Results
• Purification and structural characterization of wild-type and mutant malic enzymes
• Steady-state kinetic properties of wild-type and mutant malic enzymes
• Partial reactions catalysed by recombinant malic enzymes
• pH studies
• Chemical rescue experiments
• Discussion
• Experimental procedures
• Materials
• Cloning of pigeon liver malic enzyme cDNA
• Site-directed mutagenesis
• Expression and purification of recombinant malic enzymes
• CD measurements
• Enzyme assay
• Kinetic analysis
• Partial reaction analysis
• pH studies
• Chemical rescue
• Acknowledgements
• References
• Supplementary material