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Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase |
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| Authors: | Amit K Gandhi Faik N Musayev Samuel O Aboagye Verne Schirch |
| Institution: | a Department of Medicinal Chemistry, School of Pharmacy and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, 800 East Leigh Street, Suite 212, Richmond VA 23219, USA b Department of Natural Sciences, Virginia Union University, Richmond, VA 23220, USA c Department of Chemistry, Virginia Commonwealth University, Richmond VA 23284, USA d Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche, Università La Sapienza Roma, Italy |
| Abstract: | Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5′-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows ∼2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 Å) shows bound ATP, PL and PLP, while D235N (2.3 Å) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz·PLP·ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP. |
| Keywords: | Pyridoxal kinase Ribokinase Pyridoxal 5&prime -phosphate Crystallography Vitamin B6 Substrate inhibition Catalysis Phosphorylation |
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