Lactate dehydrogenase active site

Simulate these mutants for dogfish M4 lactate dehydrogeanse (6LDH.pdb). After energy minimization, superimpose the wild-type and mutant enzymes and construct their animated kinemage files including views for the complete molecular structures and close-ups of the L-lactate dehydrogenase active site as identified by the ProSite. 

Examination of the molecular dynamics (MD) simulation dehydrogenases with substrate and NAD(P)H at the active site shows that only one of the possible quasi-boat conformations exists (Bruice and Lightstone, 1998). The NAC structure in the lactate dehydrogenase active site is associated with the formation of the quasi-boat conformation. In this configuration the distance between the transferring hydride and pyruvate carbonyl is about 1 A shorter when the dihydropyridin ring is in the boat form than in the planar conformation. The closeness of the approach of the reactants in this pretransition state, and... 

The addition of cyanide or other nucleophiles to the C-4 position of NAD illustrates this point, as shown in reaction (1). Adduct formation becomes increasingly favorable as solvent polarity decreases. Burgner and Ray reported that lactate dehydrogenase activates NAD toward cyanide adduct formation by 100 times relative to NAD in solution (7i). This difference is attributed to stabilization of the neutral adduct in the relatively nonpolar active site. 

The addition of fumarate to the suspension of P. freudenreichii oxidizing lactate was shown to result in a partial oxidation of cytochromes b and 02 (de Vries et al., 1977). In the presence of 2H-heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of cytochrome b, lactate dehydrogenase activity was reduced by 85% if fumarate served as an electron acceptor, but only by 25% in the presence of methylene blue (in the latter case H2 is transferred to the dye without participation of cytochrome b). Keeping in mind the evidence presented above, the propionic acid fermentation can be viewed as a harmonious combination of the soluble and membrane-bound redox enzymes. The involvement of cytochrome b in anaerobic electron transport to fiimarate confirms the suggestion (Bauchop and Elsden, 1960) that there is an oxidative phosphorylation site between fumarate and succinate in propionibacteria. 

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. 

Figure 15-3 Diagrammatic structure of NAD+ and L-lactate bound into the active site of lactate dehydrogenase. See Eventhoff et al.5...

A key structural and mechanistic feature of lactate and malate dehydrogenases is the active site loop, residues 98-110 of the lactate enzyme, which was seen in the crystal structure to close over the reagents in the ternary complex.49,50 The loop has two functions it carries Arg-109, which helps to stabilize the transition state during hydride transfer and contacts around 101-103 are the main determinants of specificity. Tryptophan residues were placed in various parts of lactate dehydrogenase to monitor conformational changes during catalysis.54,59,60 Loop closure is the slowest of the motions. 

Holland, L.Z., M. McFall-Ngai, and G.N. Somero (1997). Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments Differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site. Biochemistry 36 3207-3215. 

One example of this kind of analysis is the work of Clarke and colleagues on the enzyme lactate dehydrogenase, published in 1989. Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate with NADH to form lactate (see Section 14.3). A schematic of the enzyme s active site is shown below the pyruvate is in the center ... 

Fig. 19. The active sites of lactate dehydrogenase (A) and glyceraldehyde-3-phosphate dehydrogenase (B). From the work of Rossmann and colleagues [52],...

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]...

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