Glutamate dehydrogenase, reaction catalyzed

Abass and colleagues developed an amperometric biosensor for NHA that uses the enzyme glutamate dehydrogenase to catalyze the following reaction. 

An amino acid is deaminated when it loses its nitrogen in the form of NH3 and generates an a-keto acid. It is a redox reaction using NAD+, FAD, or FMN as cofactors, and it may occur in either the mitochondria or the cytosol. The most potent l amino acid deaminase is glutamate dehydrogenase, which catalyzes the reaction... 

The major enzyme involved in the formation of ammonia in the liver, brain, muscle, and kidney is glutamate dehydrogenase, which catalyzes the reaction in which ammonia is condensed with 2-oxoglutarate to form glutamate (Sec. 15.1). Small amounts of ammonia are produced from important amine metabolites such as epinephrine, norepinephrine, and histamine via amine oxidase reactions. It is also produced in the degradation of purines and pyrimidines (Sec. 15.6) and in the small intestine from the hydrolysis of glutamine. The concentration of ammonia is regulated within narrow limits the upper limit of normal in the blood in humans is 70/tmol L-1. It is toxic to most cells at quite low concentrations hence there are specific chemical mechanisms for its removal. The reasons for ammonia toxicity are still not understood. The activity of the urea cycle in the liver maintains the concentration of ammonia in peripheral blood at 20/ molL. ... 

The amino acid specificity of glutamate dehydrogenase is known to be broad (e.g.. References 26,27). Since the reaction is reversible, the a-keto acid specificity should also be correspondingly broad. Indeed, Table I shows that glutamate dehydrogenase can catalyze the reductive amination of at least 10 a-keto acids. It should be noted that kinetic data were determined in die presence of a low ammonia concentration (5mM) in order to obtain realistic parameters for the design of optimal... 

The major enzyme involved in the formation of ammonia in the liver, brain, muscle, and kidney is glutamate dehydrogenase. It catalyzes the reaction in which anunonia is condensed with 2-oxoglutarate to form glutamate (Sec. 14.1). 

Glutamate dehydrogenase [EC 1.4.1.2] catalyzes the reaction of L-glutamate with NAD+ and water to produce a-ketoglutarate (or, 2-oxoglutarate), ammonia, and NADH. 

A three-substrate, three-product enzyme-catalyzed reaction scheme in which the three substrates (A, B, and C) and three products (P, Q, and R) can bind to and be released in any order. A number of enzymes have been reported to have this mechanism for example, adenylosuccinate synthetase , glutamate dehydrogenase, glutamine synthetase , formyltetrahydrofolate synthetase, and tubulin tyrosine ligase . See Multisubstrate Mechanisms... 

Glutamate can also be formed in yet another, albeit minor, pathway the reaction of a-ketoglutarate and NH4 to form glutamate in one step. This is catalyzed by L-glutamate dehydrogenase, an enzyme present in all organisms. Reducing power is furnished by NADPH ... 

Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the a-keto acids pyruvate, oxaloacetate, and a-keto-glutarate, respectively. These transamination reactions (Figure 20.12, and see p. 248) are the most direct of the biosynthetic pathways. Glutamate is unusual in that it can also be synthesized by the reverse of oxidative deamination, catalyzed by glutamate dehydrogenase (see p. 249). 

The reactions of Eq. 15-19 occur nonenzymatically only under the influence of strong base but dehydrogenases often catalyze similar condensations relatively rapidly and reversibly. Pyruvate inhibits lactate dehydrogenase, 2-oxoglutarate inhibits glutamate dehydrogenase, and ketones inhibit a short-chain alcohol dehydrogenase in this manner.133,693... 

While reductive animation of glutamate via glutamate synthase appears to be the major pathway for incorporation of nitrogen into amino groups, some direct animation of pyruvate and other 2-oxoacids in reactions analogous to that of glutamate dehydrogenase occurs in bacteria.105 106 Another bacterial enzyme catalyzes reversible addition of ammonia to fumarate to form aspartate (p. 685). 

What is the function of NADPH in the reactions catalyzed by glutamate dehydrogenase and glutamate synthase ... 

In fact, the a-ketoglutarate/glutamate dehydrogenase is a generally applicable method for the regeneration of NAD and NADP in laboratory scale productions. Both components involved are inexpensive and stable. Quite recently, a method for the oxidation of the reduced nicotinamide coenzymes based on bacterial NAD(P)H oxidase has been described [225], This enzyme oxidizes NADH as well as NADPH with low Km values. The product of this reaction is peroxide, which tends to deactivate enzymes, but it can be destroyed simultaneously by addition of catalase. The irreversible peroxide/catalase reaction favours the ADH catalyzed oxidation reaction, and complete conversions of this reaction type are described. 

Enzymes that catalyze redox reactions often require a coenzyme such as NAD+ or FADH2 in addition to a substrate. These are all multiple substrate enzymes. Each substrate and coenzyme will have its own Km value. The substrates for glutamate dehydrogenase, an enzyme with three substrates in both forward and reverse directions, are shown in Scheme 4.10 with their K values.9... 

Glutamate dehydrogenase catalyzes the reductive amination of the citric acid cycle intermediate a-ketoglutarate (Fig. 3a) (see Topic LI). Although the reaction is reversible, the reductant used in the biosynthetic reaction is NADPH. This enzyme is also involved in the catabolism of amino acids (see Topic M2). 

Carbamoyl phosphate synthetase, which is technically not a member of the urea cycle, catalyzes the condensation and activation of ammonia (from the oxidative deamination of glutamate by glutamate dehydrogenase Topic M2) and C02 (in the form of bicarbonate, HC03 ) to form carbamoyl phosphate. The hydrolysis of two ATP molecules makes this reaction essentially irreversible. 

---