1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD+ binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme-coenzyme complexes. 4. The secondary plots of the inhibitions with HC03-and NO3-are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH 6.0 is 20 times that with bicarbonate at pH 8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the Km values for NAD+ and NADH.Formate dehydrogenase (formate-NAD+ oxidoreductase; EC 1.2.1.2) catalyses the reaction:
NAD++HC02-÷ NADH+C02and is known to occur in animals, higher plants and bacteria. The higher-plant enzyme differs from both the animal enzyme, which is activated by ATP (Mathews & Vennesland, 1950), and the bacterial enzyme, which does not require NAD+ (Gale, 1939). Although Davison (1951) and Nason & Little (1955) have both reported the effects of various enzyme inhibitors on the activity of the plant enzyme, no kinetic investigation has so far been undertaken.The present study is an investigation into the mechanism of enzyme action by steady-state kinetics. Various enzyme-substrate/enzyme-inhibitor complexes and their possible pathways of interconversion were determined by using initialvelocity, product-inhibition and dead-end-inhibition analysis.
EXPERIMENTALEnzyme. Formate dehydrogenase was purified from Phaseolu8 aureu? (mung bean) by a combination of salt fractionation, ion-exchange, hydroxyapatite and aluminagel chromatography, and assayed as described by Peacock (1970). The molecular weight was estimated by gel filtration as 92000I10000, and the enzyme was not more than 50% pure, as determined by acrylamide-gel electrophoresis. Sub8trates. Analytical-grade ammonium formate and NaHCO3 were from British Drug Houses Ltd., Poole, Dorset, U.K.Initial-rate measurements. The reaction was followed by measuring the appearance of NADH at 340nm by using a Unicam SP. 800 spectrophotometer with an attached lOmV 'slave' recorder. The full-scale deflexion of the recorder was set to correspond to 0.1 extinction unit, and the chart speed adjusted to give recorded traces with an angle of inclination of between 30 and 600. Silica cells (4cm light-path) were filled with all the components of the reaction mixture except the enzyme, in a total volume of 9.9ml, and preincubated at 250C. The reaction was initiated by the addition of 0.1ml of suitably diluted enzyme and the cell was immediately placed in the reaction compartment (kept at 25°C). The recorded traces obtained were extrapolated to the time of addition, a...
