Lactate dehydrogenase binding site

The active center of an LDH subunit is shown schematically in Fig. 2. The peptide backbone is shown as a light blue tube. Also shown are the substrate lactate (red), the coenzyme NAD (yellow), and three amino acid side chains (Arg-109, Arg-171, and His-195 green), which are directly involved in the catalysis. A peptide loop (pink) formed by amino acid residues 98-111 is also shown. In the absence of substrate and coenzyme, this partial structure is open and allows access to the substrate binding site (not shown). In the enzyme lactate NAD"" complex shown, the peptide loop closes the active center. The catalytic cycle of lactate dehydrogenase is discussed on the next page. 

RGURE 13-16 The nucleotide binding domain of the enzyme lactate dehydrogenase, (a) The Rossmann fold is a structural motif found in the NAD-binding site of many dehydrogenases It consists of a six-stranded parallel /3 sheet and four a helices inspection reveals the arrangement to be a pair of structurally similar motifs... 

Examples of other recombinant enzymes in which an alteration using site-directed mutagenesis resulted in altered substrate binding efficiencies, rates of catalysis, or stability include carbonic anhydrase (Alexander, Nair Christianson, 1991), lactate dehydrogenase (Feeney, Clarke Holbrook, 1990), and several industrially important proteases (Wells etal., 1987 Siezenera/., 1991 Teplyakovcra/., 1992 Aehle et al., 1993 Rheinnecker et al., 1994). 

N. Bernard, K. Johnson, J. J. Holbrook, and J. Delcour, D175 discriminates between NADH and NADPH in the coenzyme binding site of Lactobacillus delbrueckii subsp. bulgaricus D-lactate dehydrogenase, Biochem. Biophys. Res. Commun. 1995, 208, 895-900. 

Semenza, G.L., B. Jiang, S.W. Leung, R. Passantino, J. Concordet, P. Maire, and A. Giallongo (1996). Hypoxia response elements in the aldolase A, eno-lase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia Inducible Factor-1. J. Biol. Chem. 271 32529-32537. 

Clarke, A.R., Atkinson, T., Holbrook, J.J. (1989) From analysis to synthesis new ligand binding sites on the lactate dehydrogenase framework, Parti. TrendsBiochem. Sci. 14, 101-105. 

Fig. 20.12. Schematic representation of the binding of nicotin-amide-adenine-dinucleotide (NAD+) to the active site in the ternary complex lactate dehydrogenase-NAD+-pyruvate. Note salt bridges N+ -H -0 between ArglOl pyrophosphate and Argl71 pyruvate [704]...

It was discovered in 1958 that anaerobically grown yeast contains a form of lactate dehydrogenase which is different from the d- and L-lac-tate cytochrome c reductases of aerobic yeast (306, 319). The enzyme has been partially purified (320, 321), and shown to contain flavin (320-322). Gel filtration studies have suggested a molecular weight of about 100,000 (320, 321). Preparations of the enzyme oxidize several d-2-hydroxyacids to the respective keto acids in a reversible manner (320). For the forward reaction, ferricyanide, 2,6-dichloroindophenol, menadione, and methylene blue have been used as electron acceptors, and for the reverse reaction leucomethyl viologen and FMNHa are effective electron donors (320). A number of L-2-hydroxyacids and 2-keto acids have been shown to be competitive inhibitors. Oxalate, cyanide, o-phenanthro-line, and EDTA are also potent inhibitors (320, 321, 323, 324). The inhibition by metal chelators develops slowly and is reversed by addition of Zn, Co, Mn +, or Fe + (320, 323-326). Substrates prevent the inhibition by chelators at concentrations considerably lower than their respective Km values (327). It has been suggested that EDTA inactivation involves the removal of a metal, most probably Zn +, from the substrate binding site of the enzyme (325, 326, 328, 329). However, others have... 

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