Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase.
Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.
Tung
P P
PP
U.S. Patent and Trade Mark Office, Arlington VA 22202, USA.
Alter
G M
GM
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005470:Fluorometry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008024:Ligands; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008958:Models, Molecular; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D009282:Naphthalenesulfonates; D010071:Oxaloacetates / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D013552:Swine; D013646:Tartronates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D008024:Ligands; D008293:Malates; D009124:Muscle Proteins; D009282:Naphthalenesulfonates; D010071:Oxaloacetates; D010446:Peptide Fragments; D013646:Tartronates; D009243:NAD; C032973:tartronic acid; C006421:1,5-I-AEDANS; C030298:malic acid; D008291:Malate Dehydrogenase