Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies

Abstract

Human liver glycerol-3-phosphate dehydrogenase (hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzymesubstrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E•NAD, and ternary E•NAD•DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain, and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains was likewise examined by double mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

Computational mechanistic study of human liver glycerol 3‐phosphate dehydrogenase using ONIOM method

“…The structure of active site including surrounding residues, NADH, and DHAP extracted from the crystal structure of ternary enzyme-substrate-cofactor the addition of ethyl ammonium cation. [7] However, no rescue was observed for the D260G upon addition of formate anion. This was attributed to the inaccessibility of Asp260 to the solvent molecules and its being located under Lys120.…”

“…Furthermore, another nearby Lys residue, Lys120, has a hydrogen bonding interaction with hydroxyl group of DHAP. Recently, Mydy et al [7] have reported high resolution X-ray crystal structures of enzyme and binary-ternary complexes of hlGPDH with NADH and DHAP. They highlighted the same conformational change upon substrate and cofactor binding as previously reported by Ou et al [2] A flexible protein loop (292-LNGQKL-297) folds over the side chain of Arg269 that interacts directly to form an ion pair with the phosphodianion of the DHAP.…”

“…Based on these results, it was concluded that the catalysis is carried out though the transition state stabilization by electrostatic interactions with polar residues. These experimental studies [2,7] provided invaluable information in the elucidation of the reaction mechanism of hlGPDH. However, there still exist ambiguities related to the roles of active site residues having catalytic importance.…”

“…These experimental studies [ 2,7 ] provided invaluable information in the elucidation of the reaction mechanism of hl GPDH. However, there still exist ambiguities related to the roles of active site residues having catalytic importance.…”

“…However, there still exist ambiguities related to the roles of active site residues having catalytic importance. Based on the crystal structure of the ternary enzyme‐NADH‐DHAP complex (Figure 3) [ 7 ] , Lys204 may not act easily as the catalytic acid due to its being below the plane of DHAP carbonyl group. However, Lys120 may also not act as a catalytic acid protonating carbonyl carbon of DHAP.…”