Dehydrogenases, Changing Cofactor Specificity

Dehydrogenases are very valuable en2ymes in biocatalysis, although one of their challenges is the use of an additional cofactor, such as NAD(P)(H). There are several advantages to dealing only with dehydrogenases that are dependent on NAD(H) but not on NADP(H)  

One successful example regarding a specificity change from NADP(H) to NAD(H) was achieved by Riebel and Hummel at the Research Center Jiilich (Jiilich, Germany) they succeeded in affecting a specificity change on the (R)-ADH of L. brevis (Hummel, 1999 Riebel, 2002). NAD(P)-specificity is conferred by a single residue in position 37 an Asp or Glu shifts the specificity to NAD, whereas a small uncharged residue such as Gly next to an Arg leads to preference for NADP over NAD. However, while the specificity now favors NAD by a factor of 300, the rate went down by a factor of 4. The properties of the wild-type enzyme and two mutants are listed in Table 10.5. 

Property Wild type Mutein 2 (R38L/K48M/A9G) Mutein C37D 

Another example of cofactor exchange was performed using a mammalian cytochrome P450, changing the specificity from NADP to NAD, which is discussed in detail Section 10.7. 

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Chen Z, Tsigelny I, Lee WR, Baker ME, Chang SH. Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+. FEBS Lett 356 1994 81-85. 

The most confusing aspect of the pathway proposed by Ochoa and his group now rests with the NAD requirement. In proceeding from L-malic acid to L-lactic acid, there is no net change in oxidation state. Yet in whole cells or cell-free extracts, the malo-lactic fermentation will not proceed in the absence of NAD. Therefore, by the proposed mechanism, one is unable to demonstrate the appearance of reduced cofactor, and the NAD specificity cannot be explained as a redox requirement. However, in the time since this mechanism was proposed, an NAD dependent enzyme (glyceraldehyde-3-phosphate dehydrogenase) has been described which requires NAD in a non-redox capacity (29), and it is possible that the same is true for the enzyme causing the malic acid-lactic acid transformation. 

The demonstration by Phillips and Laiigdon (1956) that the level of TPNH-cytochromc c reductase is increased in the livers of hyperthyroid rats and reduced in those of hypothyroid animals is of particular interest. Since the activity of this enzyme results in the production of triphospho-pyridiiie nucleotide (TPN), a cofactor required for many oxidative enzymes, this change is probably a direct reflection of the general over-all acceleration of oxidative processes, and, more specifically, of those which are TPN-linked. Decreased activity of lactic dehydrogenase (Vcstling and Knocpfelmacher, 1950) is compatible with the demonstrated ability of thyroxine to inhibit this enzyme in vitro (Wolff and Wolff, 1957 Radsma et al., 1957). Decreases in the in vivo activity of such enzymes as tyrosine oxidase, DOPA-decarboxylase, betaine-homocysteine transmethylase, and... 

Figure 37.4 Three-dimensional structure of electron-transfer flavoprotein (ETF) alone and in complex with medium-chain acyl-CoA dehydrogenase (MCAD). (A) Human ETF is a dimer of two distinct polypeptide chains, and harbours FAD and AMP cofactors (white sticks). Structure is divided in three sub-domains that are shown in roman numerals. (B) Crystallographic structure of ETF MCAD complex. ETF domain III is responsible for establishing protein-protein specific interactions. ETF domain II undergoes a dramatic conformational change upon complex formation (compare flavin position in panel A) in order to allow efleetive electron transfer to the flavin of MCAD. Structures of ETF and ETF MCAD complexes were obtained from Protein Data Bank (PDB lefv and 2A1T, respectively).

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