Glycerol-3-Phosphate Dehydrogenase: Investigation of Intricate Interactions of K120 and D260 in the Enzyme Active Site

• Main Author: Katigbak, Roberto D
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2017
• Subjects: Chemistry
• ISBN: 0355309998; 9780355309997

Glycerol-3-phosphate dehydrogenase is a key enzyme that links the glycolysis pathway to triacylglyceride synthesis. It catalyzes the reduction of dihydroxyacetone phosphate to L-glycerol 3-phosphate using NADH as cofactor. Evaluation of the kinetic parameters of K120A, D260G and K120A/D260G mutants GPDH yielded kcat values of 0.87, 0.13 and 7.1 x 10-3 s-1, respectively and a similar Km value (1.6 – 1.9 x 10-3 M) for all mutants. Using these values and comparing them with the kinetic parameter of wild type hl GPDH yielded 5.4, 6.6 and 8.3 kcal/mol transition state destabilization for K120A, D260G and K120/D260G mutants. The same Km value accounts for interactions lost when either the side chain amine is changed (K120A mutant) or is not in proper orientation (D260G and K120A/D260G mutants). Decreased kcat values suggests the effect of mutated residues is either participating in catalysis by K120 side chain, which can act as a general acid, or disruption of intricate interactions of K120 and D260 with adjacent residues, especially K204. Chemical rescue of the K120A activity is possible by addition of exogenous ammonium cations, with 3.4, 3.0 and 1.8 kcal/mol stabilization of transition state by CH3NH3Cl, CH 3CH2NH3Cl and NH4Cl respectively. Using CH3NH3Cl at low concentrations, an increase in kcat and decrease in Km was observed for K120A GPDH with almost equal contribution with transition state stabilization. Weak inhibition was observed for D260G by formate and no rescue was seen for K120A/D260G by formate and methylammonium chloride. Overall, it is observed that clipping of the side chains of K120 and D260 to GPDH mainly disrupts the complex interactions among these side chains, adjacent residues K204 and T264, and substrate DHAP.