Hydride transfer coenzyme activation

Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

Distinct coenzymes are required in biological systems because both catabolic and anabolic pathways may exist within a single compartment of a cell. The nicotinamide coenzymes catalyze direct hydride transfer (from NAD(P)H or to NAD(P)+) to or from a substrate or other cofactors active in oxidation-reduction pathways, thus acting as two-electron carriers. Chemical models have provided... 

Nicotinamide adenine dinucleotide is a coenzyme which is only loosely bound to the active site of the enzymes with which it interacts and is free therefore, to dissociate from the enzyme during the catalytic cycle. The role of the dehydrogenase enzyme is to bring together the substrate and the NAD+ in the correct orientation for the two to react. These NAD+-dependent enzymes are known as dehydrogenases. They work in conjunction with NAD+ to oxidise substrates by the transfer of 1H+ and 2e from the substrate to the 4-position of the nicotinamide ring of the NAD+ (see Fig. 2.1). The overall reaction is the equivalent of a hydride transfer and is commonly referred to as such. NAD+-dependent enzymes are primarily involved in respiration (NAD+ occurs in significant amounts in mitochondria), whereas, NADP+-dependent coenzymes are primarily involved in the transfer of electrons from intermediates in catabolism. 

Lactate dehydrogenase is a pyridine nucleotide oxidoreductase, a tetramer of 140 kD molecular weight, which has been extensively investigated (Bloxham et al., 1975 Eventoff et al., 1977). It catalyses the reversible oxidation of L-lactate to pyruvate using NAD+ as a coenzyme. The reaction scheme with a view of the active site with bound substrate and essential amino-acid side chains are depicted in Equation (3) and in Figure 17. The probable reaction mechanism, involving proton and hydride transfers,... 

Activation of the coenzyme for hydride transfer involves a complex interplay among a number of factors. These include distortion of the nicotinamide/dihy-dronicotinamide ring, solvation, direct electrostatic interactions, conformation of the side chain at C-3, conformation around the glycosyl bond, and inductive effects directed at the sugar moiety. The following sections will provide a discussion of these interactions and their potential mechanistic impact. 

Kinetic studies of reversible inhibition by substrate analogs give evidence of the mode of action of the inhibitor and the types of enzyme-inhibitor complex formed, and estimates of their dissociation constants. The complexes may be isolated and sometimes crystallized. Studies of the stabilities, optical properties, and structures of ternary complexes of enzymes, coenzymes, and substrate analog in particular, as stable models of the catalytically active ternary complexes or of the transition state for hydride transfer (61,79,109,115-117), can only be touched upon here there is direct evidence with several enzymes that the binding of coenzymes is firmer in such complexes than in their binary complexes (85,93,118), which supports the indirect, kinetic evidence already mentioned for a similar stabilization in active ternary complexes. 

The hydride transfer step is a direct transfer between substrate and coenzyme. The suggestion by Schellenberg (363,369) that tryptophan mediates this hydride transfer in the yeast enzyme has been shown chemically not to be valid for both yeast and liver enzyme (93,370-373). Furthermore, the tertiary structure shows that the two tryptophans of the LADH subunit are both approximately 20 A away from the active site... 

Oxidation-reduction coenzymes follow the same principles as activation-transfer coenzymes, except that they do not form covalent bonds with the substrate. Each coenzyme has a unique functional group that accepts and donates electrons and is specific for the form of electrons it transfers (e.g., hydride ions, hydrogen atoms, oxygen). A different portion of the coenzyme binds the enzyme. Like activation-transfer coenzymes, oxidation-reduction coenzymes are not good catalysts without participation from amino acid side chains on the enzyme. 

In principle, many targetor moieties are possible for a general system of this kind [119, 122-125], but the one based on the 1,4-dihydrotrigonelline trigonelline (coffearine) system, where the lipophilic 1,4-dihydro form (T) is converted in vivo to the hydrophilic quaternary form (T ), proved the most useful. This conversion takes place easily everywhere in the body since it is closely related to the ubiquitous NADH NAD coenzyme system associated with numerous oxidoreductases and cellular respiration [126, 127]. Since oxidation takes place with direct hydride transfer [128] and without generating highly active or reactive radical intermediates, it provides a non-toxic targetor system [129]. Furthermore, since for small quaternary pyridinium ions rapid elimination from the brain, probably due to involvement of an active transport... 

Reduced nicotinamide-adenine dinucleotide (NADH) plays a vital role in the reduction of oxygen in the respiratory chain [139]. The biological activity of NADH and oxidized nicotinamideadenine dinucleotide (NAD ) is based on the ability of the nicotinamide group to undergo reversible oxidation-reduction reactions, where a hydride equivalent transfers between a pyridine nucleus in the coenzymes and a substrate (Scheme 29a). The prototype of the reaction is formulated by a simple process where a hydride equivalent transfers from an allylic position to an unsaturated bond (Scheme 29b). No bonds form between the n bonds where electrons delocalize or where the frontier orbitals localize. The simplified formula can be compared with the ene reaction of propene (Scheme 29c), where a bond forms between the n bonds. 

Enantioselective oxidation of racemic alcohols as well as reduction of racemic ketones and aldehydes have been widely applied to obtain optically active alcohols.25 27 The enzymes catalyzing these reactions are alcohol dehydrogenase, oxidases, and reductases etc. Coenzymes (NADH, NADPH, flavine etc) are usually necessary for theses enzymes. For example, for the oxidation of alcohols, NAD(P)+ are used. The hydride removed from the substrate is transferred to the coenzyme bound in the enzyme, as shown in Figure 24. There are four stereochemical patterns, but only three types of the enzymes are known. 

Reaction evidently involves formation of the S-thiohemiacetal with the super-reactive Cys-149. His-176 is thought to activate Cys-149 and to facilitate proton removal (the oxidation step being transfer of hydride to the coenzyme, with formation of the thioacyl intermediate). Thr-179 and residue 181 (e.g., Thr or Asn) probably interact with the 3-phosphate, while Ser-148 can possibly form hydrogen bonds with the C-2 hydroxyl and with the inorganic phosphate (Fig. 19B) [52]. Thr-208 or Arg-231 may also bind the inorganic phosphate. Nucleophilic attack by the phosphate on C-l, with rupture of the C-S bond, gives 1,3-diphosphoglycerate. 

Several different amino acid side chains can act as nucleophiles in enzyme catalysis. The most powerful nucleophile is the thiol side chain of cysteine, which can be deproto-nated to form the even more nucleophilic thiolate anion. One example in which cysteine is used as a nucleophile is the enzyme glyceraldehyde 3-phosphate dehydrogenase, which uses the redox coenzyme NAD+. As shown in Fig. 10, the aldehyde substrate is attacked by an active site cysteine, Cys-149, to form a hemi-thioketal intermediate, which transfers hydride to NAD+ to form an oxidized thioester intermediate (7). Attack of phosphate anion generates an energy-rich intermediate 3-phosphoglycerate. 

Unanticipated developments help to put known facts into place. Results from biochemistry drove home to organic chemists the message that it was not a chemical rarity for carbon-hydrogen bonds to be sources of hydride equivalents. Westheimer, Vennesland et al. established beyond doubt that in a redox reaction mediated by the coenzyme couple NAD(P)+/NAD(P)H the carbon-hydrogen bond of ethanol could serve directly as a hydride donor to an electron-deficient carbon of a pyridinium ion, and that this hydride equivalent could in turn be donated directly to the electropositive carbon of a carbonyl group. Thus the hydride donor capacities of carbon are also part and parcel of life. All this can occur under physiological conditions with the help of an enzyme, which somehow activates these reactants. The sequence is illustrated schematically in equation (1). In either direction hydride is transferred from carbon to carbon. 

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