1 Kinetics and Mechanism of Nicotinamide-Nucleotid-Linked Dehydrogenases

doi:10.1016/S1874-6047(08)60209-7

The Enzymes

Volume 11, 1975, Pages 1-60

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Nicotinamide-nucleotide-linked dehydrogenases provide much of the original stimulus for the necessary extension of kinetic theory already developed for one-substrate and hydrolytic enzymes. This was partly because of the convenience and precision with which rates can be measured by means of the light absorption or fluorescence emission of the reduced coenzymes and because of the changes of these properties, which accompany the binding of reduced coenzymes to many dehydrogenases. Knowledge of the static structure of an enzyme puts the same advantageous position as the chemist studying the mechanism of a reaction between simpler compounds of known structure. The nicotinamide nucleotides are coenzymes, and not prosthetic groups, and can be considered as substrates from the kinetic point of view. They also form stable and reversible compounds with dehydrogenases. It is reasonable to suppose that the enzyme-coenzyme compounds are intermediates in the overall catalytic reaction. A compulsory-order mechanism with the coenzyme combining first with the enzyme and dissociating last, has been established for a few dehydrogenases.

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Cited by (120)

Purification and characterization of alanine dehydrogenase from a marine bacterium, Vibrio proteolyticus
2003, Journal of Molecular Catalysis B: Enzymatic
Alanine dehydrogenase of Vibrio proteolyticus DSM 30189 shows high activity toward β-hydroxypyruvate, and the enzyme is applicable to the production of l-serine. We have cloned the enzyme gene from the bacterium into Escherichia coli TG1 with a vector plasmid, pUC118. The enzyme was overproduced by the transformed cells and purified to homogeneity with a yield of 46%. The molecular mass of the enzyme was about 230 kDa and consisted of six identical subunits. The enzyme showed broad specificity toward α-keto acids in the reductive amination. The relative activities of the enzyme for pyruvate, β-fluoropyruvate, and β-hydroxypyruvate were 100%, 74%, and 54%, respectively. The enzyme retained more than 90% of the activity after incubation at 65 °C for 60 min in the presence of 2.0 M NaCl, but 98% of its original activity was lost in the absence of NaCl. RbCl, as well as NaCl, significantly stabilized the enzyme. On the other hand, LiCl and KCl were not as effective as stabilizers such as RbCl and NaCl.
Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase
2002, Journal of Biological Chemistry
Citation Excerpt :
For saturation studies, wild-type GDH (100 μg) in 10 mm Tris acetate, pH 8.0, containing 12 mmglutarate was incubated with various concentrations of [32P]2N3NAD+ in Eppendorf tubes for 5 min. Glutarate was included in the reaction mixture because it has been reported by equilibrium dialysis and initial rates that glutarate makes NAD+ bind to GDH more tightly (39-41). For competition studies, 100 μg of enzyme was incubated with various concentrations of NAD+ for 10 min in the same buffer before the addition of 100 μm[32P]2N3NAD+ and then incubated with the photoprobe for 5 min as described above.
Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [³²P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD⁺ binding site within human GDH encoded by the synthetic human GDH gene and expressed inEscherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD⁺ during photolysis, demonstrating a selectivity of the photoprobe for the NAD⁺ binding site. Amino acid sequencing and compositional analysis identified Glu²⁷⁹ as the site of photoinsertion into human GDH, suggesting that Glu²⁷⁹ is located at or near the NAD⁺ binding site. The importance of the Glu²⁷⁹ residue in the binding of NAD⁺ was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu²⁷⁹ has no effects on the expression or stability of the different mutants. The K _mvalues for NAD⁺ were 10–14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V _maxvalues were similar for wild-type and mutant GDHs. The efficiency (k _cat/K _m) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K _m values for NAD⁺. In contrast to the K _m values for NAD⁺, wild-type and mutant GDHs show similarK _m values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu²⁷⁹ is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu²⁷⁹ plays an important role for efficient binding of NAD⁺ to human GDH.
The structure of apo human glutamate dehydrogenase details subunit communication and allostery
2002, Journal of Molecular Biology
The structure of human glutamate dehydrogenase (GDH) has been determined in the absence of active site and regulatory ligands. Compared to the structures of bovine GDH that were complexed with coenzyme and substrate, the NAD binding domain is rotated away from the glutamate-binding domain. The electron density of this domain is more disordered the further it is from the pivot helix. Mass spectrometry results suggest that this is likely due to the apo form being more dynamic than the closed form. The antenna undergoes significant conformational changes as the catalytic cleft opens. The ascending helix in the antenna moves in a clockwise manner and the helix in the descending strand contracts in a manner akin to the relaxation of an extended spring. A number of spontaneous mutations in this antenna region cause the hyperinsulinism/hyperammonemia syndrome by decreasing GDH sensitivity to the inhibitor, GTP. Since these residues do not directly contact the bound GTP, the conformational changes in the antenna are apparently crucial to GTP inhibition. In the open conformation, the GTP binding site is distorted such that it can no longer bind GTP. In contrast, ADP binding benefits by the opening of the catalytic cleft since R463 on the pivot helix is pushed into contact distance with the β-phosphate of ADP. These results support the previous proposal that purines regulate GDH activity by altering the dynamics of the NAD binding domain. Finally, a possible structural mechanism for negative cooperativity is presented.
Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation
2001, Journal of Molecular Biology
Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. However, only animal GDH utilizes both NAD(H) or NADP(H) with comparable efficacy and exhibits a complex pattern of allosteric inhibition by a wide variety of small molecules. The major allosteric inhibitors are GTP and NADH and the two main allosteric activators are ADP and NAD⁺. The structures presented here have refined and modified the previous structural model of allosteric regulation inferred from the original boGDH·NADH·GLU·GTP complex. The boGDH·NAD⁺·α-KG complex structure clearly demonstrates that the second coenzyme-binding site lies directly under the “pivot helix” of the NAD⁺ binding domain. In this complex, phosphates are observed to occupy the inhibitory GTP site and may be responsible for the previously observed structural stabilization by polyanions. The boGDH·NADPH·GLU·GTP complex shows the location of the additional phosphate on the active site coenzyme molecule and the GTP molecule bound to the GTP inhibitory site. As expected, since NADPH does not bind well to the second coenzyme site, no evidence of a bound molecule is observed at the second coenzyme site under the pivot helix. Therefore, these results suggest that the inhibitory GTP site is as previously identified. However, ADP, NAD⁺, and NADH all bind under the pivot helix, but a second GTP molecule does not. Kinetic analysis of a hyperinsulinism/hyperammonemia mutant strongly suggests that ATP can inhibit the reaction by binding to the GTP site. Finally, the fact that NADH, NAD⁺, and ADP all bind to the same site requires a re-analysis of the previous models for NADH inhibition.
Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase
2001, Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Citation Excerpt :
Certainly, these observations are not consistent with a strictly ordered sequential binding mechanism in which one type of ligand must bind before the other. Such a cofactor-first ordered mechanism is frequently invoked for dehydrogenase [32]. In the case of malate dehydrogenase, evidence of an ordered mechanism has been reported for microbial and mammalian enzymes (i.e. [10,11,33–36]).
Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme’s interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme’s structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme’s active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label’s fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.
Substrate channeling
1999, Methods: A Companion to Methods in Enzymology
Substrate channeling is the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing with the bulk phase. This process is equivalent to a microcompartmentation of the intermediate, although classic diffusion occurs simultaneously to varying extents in many of these cases. This microcompartmentation and other factors of channeling provide many potential biological advantages. Extensive examples of channeling can be found in the cited reviews. The choice of methods to detect and characterize substrate channeling depends extensively on the type of enzyme associations involved, the constants of the system, and, to some extent, the mechanism of channeling. Thus it is important to distinguish stable, dynamic, and catalytically induced enzyme associations as well as recognize different mechanisms of substrate channeling. We discuss the principles, experimental details, and limitations and precautions of five rather general methods. These use measurements of transient times, isotope dilution or enhancement, competing reaction effects, enzyme buffering kinetics, and transient-state kinetics. These encompass methods applicable to studies in vitro, in situ, and in vivo. None of these methods is applicable to all systems. They are also susceptible to artifacts without proper attention to precautions. Transient-state kinetic methods clearly excel in elucidating molecular mechanisms of channeling. However, they are often not the best method for initial detection and characterization of the process and they are not applicable to many complex systems. Several other methods that have been successful in indicating substrate channeling are briefly described.

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1 Kinetics and Mechanism of Nicotinamide-Nucleotid-Linked Dehydrogenases

Publisher Summary

JBC

BBA

“The Enzymes,”

“The Enzymes,”

JBC

JBC

JBC

JBC

JBC

JBC

JBC

JBC

JBC

JBC

JBC

JBC

JBC

BBA

JBC

ABB

JBC

JBC

JBC

JBC

BBA

BBA

BBA

BBA

Acta Chem. Scand.

Acta Chem. Scand.

Acta Chem. Scand.

JACS

Acta Chem. Scand.

JACS

Acta Chem. Scand.

BJ

BJ

Acta Chem. Scand.

Compr. Biochem.

“Enzymes.”

BJ

BJ

Acta Chem. Scand.

Proc. Nat. Acad. Sci. US.

Proc. Nat. Acad. Sci. U. S.

Nature (London)

Biochemistry