Active site malate dehydrogenase

FIGURE 20,20 (a) The structure of malate dehydrogenase, (b) The active site of malate dehydrogenase. Malate is shown in red NAD" is blue. 

Linked oxidation and decarboxylation. Metabolic pathways often make use of oxidation of a (3-hydroxy acid to a (3-oxoacid followed by decarboxylation in the active site of the same enzyme. An example is conversion of L-malate to pyruvate (Eq. 13-45). The Mg2+ or Mn2+-dependent decarboxylating malic dehydrogenase that catalyzes the reaction is usually called the malic enzyme. It is found in most organisms.237-240 While a concerted decarboxylation and dehydrogenation may sometimes occur,241-242 the enzymes of this group appear usually to operate with bound oxoacid intermediates as in Eq. 13-45. 

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. 

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

A second type of EMIT has been developed using the enzyme malate dehydrogenase as the enzymatic label. Research has shown that thyroxine competitively inhibits malate dehydrogenase. A conjugate prepared with thyroxine covalently bound close to the enzyme s active site shows very low specific activity that can be restored by binding of the thyroxine to arcP -thyroxine antibody. In this very specific assay for thyroxine, enzyme activity increases upon antibody binding, so that in a competitive assay for free thyroxine, activity decreases with increasing free thyroxine concentration. 

Figure 9.8. The malate dehydrogenase dimer, indicating the location of the active sites in each protein plus the dimer interface. Malate dehydrogenase demonstrates substrate inhibition that has been attributed to subunit interactions and allosteric regulation by citrate, although the crystal structure of the protein reveals the absence of a separate allosteric site for citrate. See color insert.

ATP, ADP, and AMP inhibit the malate dehydrogenase from E. coli but in an allosteric manner 86). Sanwal has suggested that in the case of MDH from E. coli there is an additional (allosteric) site for NADH and the adenine nucleotides affect activity by binding at this site (s) 86). 

In this reaction, malate and NAD diffuse into the active site of malate dehydrogenase. Here NAD accepts two electrons from malate oxaloacetate and NADH then diffuse out of the active site. The reduced NADH must then be returned to its NAD form. For each catalytic cycle, a new NAD molecule is needed if the reaction is to occur thus, stoichiometric quantities of the cosubstrate are needed. The reduced form of this coenzyme (NADH) is converted back to the oxidized form (NAD ) via a number of simultaneously occurring processes in the cell, and the regenerated NAD can then participate in another round of catalysis. 

Kirk Wright S, Viola RE (2001) Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine. J Biol Chem 276(33) 31151-311551 Klein MD, Danger R (1986) ImmobilizEd enzymes in clinical medicine an emeiging approach to new drug therapies. TIBTECH 4 179-186... 

The active site of lactate dehydrogenase could be redesigned to convert it to a malate dehydrogenase. (It was already known that the tertiary structures of the two enzymes were similar.) To take account of the increased... 

Electrostatic channeling of a substrate between two enzymatic active sites has recently been studied by Brownian dynamics for citric acid cycle enzymes malate dehydrogenase and citrate synthase,and in the bifunctional enzyme... 

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