Hydrogenases substrate specificity

Both [NiFe] and [Fe] hydrogenases are often complexed to other redox-enzyme modules with various substrate specificities (dependent on the metabolism of the microorganism). 

This enzyme [EC 1.6.1.1] (also known as NAD(P)+ trans-hydrogenase (B-specific), pyridine nucleotide transhy-drogenase, and nicotinamide nucleotide transhydro-genase) catalyzes the reversible reaction of NADPH with NAD+ to produce NADP+ and NADH. This FAD-dependent enzyme is B-specific with respect to both pyridine coenzymes. In addition, deamino coenzymes will also serve as substrates. 

Hydrogenases, like most enzymes, are extraordinary in their specificity. They catalyse a specific reaction with a particular set of substrates, and produce particular products. The reaction can go backwards and forwards many times without producing unwanted by-products. They do not, for example, reduce CO2 to CO, which would... 

Proteins and antibodies are natural substrates for affinity columns because of the nature of the enzyme recognition site and the antibody-antigen interaction sites. They have a three-dimensional shape and electrical charge distributions that interact with only specific molecules or types of molecules. Once these substrate sites are identified, molecules can be isolated or synthesized with the key characteristics and used to build affinity supports. These substrates are often bound to a 6-carbon spacer so that they protrude farther away from the packing surface toward the mobile phase and are therefore more available. Certain natural and synthetic dyes have been found to serve as substrate mimics for a class of enzymes call hydrogenases and have been used to build affinity columns for their purification. 

He presented the design of the hydrogenases and pointed out that one of the major problems is that these systems are subject to dynamic equilibria. All of the substrates, electrons included, are transported in and out of the active site in a very specific way based on numerous studies. 

Clostridial ferredoxin was discovered by virtue of its requirement in the oxidation of pyruvate by the clostridial species specifically involved in a nitrogen-fixing system (44). We can now interpret the requirement for pyruvate oxidation as a reaction leading to the formation of reduced ferredoxin, which in turn acted in the reduction of the various substrates, including N2 (45). But there are many other substrates which can be reduced by the reduced ferredoxin—i.e., in the presence of the appropriate enzyme. For example, nitrite may be reduced to ammonia. Reduced ferredoxin can be formed by nonphotosynthetic bacteria by reactions other than puruvate oxidation it can also be formed by the hydrogenase, or by the oxidation of a purine. 

Methotrexate inhibits specimens of dihydrofolate hydrogenase isolated from both mammalian and bacterial cells, although it cannot normally penetrate into the latter (Nichol and Welch, 1950 Werkheiser, 1963). The action of methotrexate is highly specific 50 per cent inhibition is effected by a io M concentration of the agent which has hardly any effect on any other enzyme. This inhibitor is boimd to the enzyme about 10 more tightly than the substrate, a most unusual effect (see Section 9.1), and hence is one of the least reversible of reversible inhibitors. The differences between its action on dihydrofolate inhibitors from different species is not large (contrast with the diaminopyrimidines, below) (Werkheiser,... 

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