Lactate dehydrogenase catalysis

Isotope effects have also been applied extensively to studies of NAD+/NADP+-linked dehydrogenases. We typically treat these enzymes as systems whose catalytic rates are limited by product release. Nonetheless, Palm clearly demonstrated a primary tritium kinetic isotope effect on lactate dehydrogenase catalysis, a finding that indicated that the hydride transfer step is rate-contributing. Plapp s laboratory later demonstrated that liver alcohol dehydrogenase has an intrinsic /ch//cd isotope effect of 5.2 with ethanol and an intrinsic /ch//cd isotope effect of 3-6-4.3 with benzyl alcohol. Moreover, Klin-man reported the following intrinsic isotope effects in the reduction of p-substituted benzaldehydes by yeast alcohol dehydrogenase kn/ko for p-Br-benzaldehyde = 3.5 kulki) for p-Cl-benzaldehyde = 3.3 kulk for p-H-benzaldehyde = 3.0 kulk for p-CHs-benzaldehyde = 5.4 and kn/ko for p-CHsO-benzaldehyde = 3.4. 

Proton transfers are particularly common. This acid-base catalysis by enzymes is much more effective than the exchange of protons between acids and bases in solution. In many cases, chemical groups are temporarily bound covalently to the amino acid residues of the enzyme or to coenzymes during the catalytic cycle. This effect is referred to as covalent catalysis (see the transaminases, for example p. 178). The principles of enzyme catalysis sketched out here are discussed in greater detail on p. 100 using the example of lactate dehydrogenase. 

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. 

The principles of enzyme catalysis discussed on p. 90 can be illustrated using the reaction mechanism of lactate dehydrogenase (LDH) as an example. 

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). 

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. 

The relative activity of the fragmentary enzyme to the wild-type enzyme was much higher than that of the clipped to the native DadB enzyme. This was explained by the assumption that the thermostable enzyme has more extensive hydrophobic interdomain interactions than the DadB enzyme with less thermostability.12 The importance of hydrophobic interdomain interactions for catalysis was pointed out by studies on lactate dehydrogenase.23,24 ... 

Deng, H., J. Zheng, A. Clarke, J.J. Holbrook, R. Callender, and J.W. Burgner II (1994). Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme-substrate complex. Biochemistry 33 2297-2305. 

Fig. 4.2. The oxidation mechanisms of lactate by sulfate in the sulfate-reducing bacteria of Desulfovibrio genus. Circled numbers 1, lactate dehydrogenase (cytochrome c-553) 2, pyruvate-ferredoxin 2-oxidoreductase (CoA-acetylating) 3, phosphate acetyltransferase 4, acetate kinase 5, sulfate adenylyltransferase 6, adenylylsulfate reductase 7, sulfite reductase 8, adenylate kinase. ATP adenosine 5 -triphosphate is also biosynthesized by the catalysis of ATP synthase using the energy liberated by the electron transfer around this part...

R. H. Callender, Toward an Understanding of the Role of Dynamics on Enzymatic Catalysis in Lactate Dehydrogenase, Biochemistry, 41, 3353-3363 (2002). 

Gulotta, M., Deng, H., Dyer, R. B., Callender, R. H. (2002) Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase, Biochemistry 41, 3353-3363. 

The catalysis of the oxidation of aldehydes to carboxylates by alcohol dehydrogenases raises questions regarding the function of the active site thiols found in most aldehyde dehydrogenases. Clearly a free thiol is not mechanistically essential for aldehyde oxidation. For example, pig heart lactate dehydrogenase catalyzes the facile oxidation of glyoxalate to oxalate (71), glucose-6-... 

The catalysis of oxidation-reduction reactions is carried out by a class of enzymes called oxidoreductases. A subclass of oxidore-ductases is given the common name dehydrogenases (such as lactate dehydrogenase), because they transfer hydrogen (hydrogen atoms or hydride atoms) from the substrate to an electron-accepting coenzyme, such as NAD. ... 

Lactate dehydrogenase was subjected to extensive enzyme engineering studies using site-directed mutagenesis to explore the roles of a number of side chains in catalysis, to examine the nature of the rate-limiting step... 

Evans SA Shore JD (1980) The role of zinc-bound water in liver alcohol dehydrogenase catalysis. J Biol Chem 255 1509-1514 Eventoff W, Rossmann MG, Taylor SS, Torff H-J, Meyer H, Keil W, Kiltz H-H (1977) Structural adaptations of lactate dehydrogenase isozymes. Proc Natl Acad Sci USA 74 2677-2681 Fisher HF, Conn EE, Vennesland B, Westheimer FH (1953) The enzymatic transfer of hydrogen I. The reaction catalyzed by alcohol... 

Holbrook JJ, Stinson RA (1973) The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase. Biochem J 131 739-748 Hoshide F, Ohi S, Baba N, Oda J, Inouye Y (1982) Asymmetric reduction with bis(NADH) model compounds. Agric Biol Chem 46 2173-2175 Hoshide F, Baba N, Oda J, Inouye Y (1983) Asymmetric reduction by an NADH model compound with L-prolinamide in the N1 substituent. Agric Biol Chem 47 2141-2143... 

L-lactate Lactate dehydrogenase covalently linked to carboxylic functionalized sol-gel Bio-catalysis [226]... 

In chapter S the phenomenon of on enzyme equilibria is discussed with examples. This refers to the fact that the equilibrium between enzyme-substrate and enzyme-product complexes is often near unity, even if the overall equilibrium constant for the interconversion of free substrate to free product is a large number. This does not contradict the statement that enzymes (or catalysts in general) do not affect equilibrium constants of reactions. It has to be remembered that this definition of catalysis only applies to the equilibrium between free substrates and products. An example, which illustrates this in terms of the Haldane relation, is heart lactate dehydrogenase. By the methods discussed in section 5.1 it was shown that the equilibrium constant for the two complexes... 

The specificities of the enzymes are also nicely explained The enantiomers of the substrates of L-lactate and D-glyceraldehyde 3-phosphate dehydrogenases cannot be productively bound the hydrophobic pocket of alcohol dehydrogenase will not bind the charged side chains of lactate etc. However, we do not know if conformational changes occur during catalysis or if there is strain. 

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