Substrate and cofactor specificity and selective inhibition of lactate dehydrogenase from the malarial parasite P. falciparum.
Lactate dehydrogenase from the malarial parasite Plasmodium falciparum has many amino acid residues that are unique compared to any other known lactate dehydrogenase. This includes residues that define the substrate and cofactor binding sites. Nevertheless, parasite lactate dehydrogenase exhibits high specificity for pyruvic acid, even more restricted than the specificity of human lactate dehydrogenases M4 and H4. Parasite lactate dehydrogenase exhibits high catalytic efficiency in the reduction of pyruvate, kcat/Km = 9.0 x 10(8) min(-1) M(-1). Parasite lactate dehydrogenase also exhibits similar cofactor specificity to the human isoforms in the oxidation of L-lactate with NAD+ and with a series of NAD+ analogs, suggesting a similar cofactor binding environment in spite of the numerous amino acid differences. Parasite lactate dehydrogenase exhibits an enhanced kcat with the analog 3-acetylpyridine adenine dinucleotide (APAD+) whereas the human isoforms exhibit a lower kcat. This differential response to APAD+ provides the kinetic basis for the enzyme-based detection of malarial parasites. A series of inhibitors structurally related to the natural product gossypol were shown to be competitive inhibitors of the binding of NADH. Slight changes in structure produced marked changes in selectivity of inhibition of lactate dehydrogenase. 7-p-Trifluoromethylbenzyl-8-deoxyhemigossylic acid inhibited parasite lactate dehydrogenase, Ki = 0.2 microM, which was 65- and 400-fold tighter binding compared to the M4 and H4 isoforms of human lactate dehydrogenase. The results suggest that the cofactor site of parasite lactate dehydrogenase may be a potential target for structure-based drug design.
Gomez
M S
MS
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Piper
R C
RC
Hunsaker
L A
LA
Royer
R E
RE
Deck
L M
LM
Makler
M T
MT
Vander Jagt
D L
DL
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006072:Gossypol / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D006072:Gossypol