Spectrophotometric Identification of an Active Site-specific Acyl Glyceraldehyde 3-Phosphate Dehydrogenase

Malhotra, O.P.; Bernhard, Sidney A.

doi:10.1016/s0021-9258(19)56979-4

Cited by 151 publications

(56 citation statements)

References 33 publications

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“…Chromophoric substrate analogues have been used profitably to investigate the mechanisms of a number of enzyme systems (Bernhard et al, 1965;Hinkle & Kirsch, 1970;Malhatra & Bernhard, 1968;Dunn & Hutchinson, 1973) and to detect any spectral intermediates that may occur. The p-(A,A-dimethylamino)phenyl group has been valuable as a chromophoric substrate analogue to investigate alcohol dehydrogenase-substrate interactions by both rapid reaction and spectral techniques (Dunn & Hutchinson, 1973;Callendar et al, 1988;Dunn et al, 1975;Anglis et al, 1977;Jagodzinski et al, 1977).…”

mentioning

confidence: 99%

Spectral and kinetic studies of imine product formation in the oxidation of p-(N,N-dimethylamino)benzylamine analogs by monoamine oxidase B

Edmondson¹,

Bhattacharyya²,

Walker³

1993

Biochemistry

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The oxidative deamination of p-(N,N-dimethylamino)benzylamine and N-methyl-p-(N,N-dimethylamino)benzylamine by bovine liver monoamine oxidase B has been investigated by absorption spectral, steady-state, and stopped-flow kinetic studies. An absorbing intermediate with a maximum at 390 nm is observed with either analogue in turnover experiments at neutral pH and is identified as due to the formation of protonated imine as the initial product. p-(N,N-Dimethylamino)benzaldehyde is the final product formed from either substrate analogue. Anaerobic stopped-flow measurements show N-methyl-p-(N,N-dimethylamino)benzylamine to reduce enzyme-bound flavin with a limiting rate of 1.8 s-1 concurrent with the appearance of a 390-nm absorption due to protonated imine product with a limiting rate of 1.7 s-1. Both observed rates are somewhat faster than catalytic turnover (1.5 s-1). Under anaerobic conditions, the decay of protonated N-methyl-p-(N,N-dimethylamino)benzenimine is much slower than turnover (k = 4.8 x 10(4) s-1). p-(N,N-Dimethylamino)benzylamine reduces the enzyme with a limiting rate of 2.1 s-1, which is faster than catalytic turnover (1.2 s-1). Protonated imine formation is also observed with this substrate with an apparent limiting rate of 1.3 s-1. The decay of the protonated p-(N,N-dimethylamino)benzenimine absorbance is slower than catalytic turnover but faster than the rate of aldehyde formation under anaerobic conditions. Deuterium kinetic isotope effect values of approximately 10 are observed both for flavin reduction and for protonated imine formation. No isotope effect is observed for the rate of imine decay.(ABSTRACT TRUNCATED AT 250 WORDS)

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mentioning

confidence: 99%

Spectral and kinetic studies of imine product formation in the oxidation of p-(N,N-dimethylamino)benzylamine analogs by monoamine oxidase B

Edmondson¹,

Bhattacharyya²,

Walker³

1993

Biochemistry

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“…Different absorption spectra of crystalline /?-2-furylacryloyl-glyceraldehyde-3phosphate dehydrogenase, in the absence and in the presence of saturating concentrations of NAD+, have been shown to be associated, respectively, with the inert and the catalytically active states of the acyl-enzyme (Berni et al 1977 ;Vas et al 1979), as in solution (Malhotra & Bernhard 1968. Furtherm ore, activation of the acyl bond by NAD+ appeared to be mediated by a protein isomerization (Mozzarelli et al 1982).…”

Section: Discussionmentioning

confidence: 98%

“…Glyceraldehyde-3-phosphate dehydrogenase The use of chromophoric reagents to prepare covalent enzyme derivatives th a t are m etastable analogues of true catalytic intermediates was extended by Sidney Bernhard and his co-workers to investigate some aspects of the catalytic and regulatory mechanism of the tetram eric enzyme glyceraldehyde-3-phosphate dehydrogenase (Malhotra & Bernhard 1968. In solution, the reaction of /?-(2furyl)acryloyl phosphate with NAD+-saturated muscle or yeast glyceraldehyde-3phosphate dehydrogenase to form the /?-2-furylacryloyl-enzyme exhibits negative cooperativity, or ' half-of-the-sites ' reactivity.…”

Section: (B)mentioning

confidence: 99%

Time course of chemical and structural events in protein crystals measured by microspectrophotometry

Rossi

Mozzarelli

Peracchi

et al. 1992

Phil. Trans. R. Soc. Lond. A

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The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.

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“…NAD+ and glyceraldehyde 3-phosphate were assayed enzymatically by the procedure of Ferdinand (1964). |8-(2-Furyl)acryloyl phosphate barium salt was prepared and solubilized as described earlier (Malhotra & Bernhard, 1968). Its concentration was assayed spectrophotometrically (emnm = 2.62 X 104 M""1 cm"1).…”

Section: Methodsmentioning

confidence: 99%

Role of nicotinamide adenine dinucleotide as an effector in the formation and reactions of acyl-glyceraldehyde-3-phosphate dehydrogenase

Malhotra¹,

Bernhard²

1981

Biochemistry

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The equilibrium spectral and reactivity properties of a chromophoric acylglyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) have been previously reported. Transient studies of these properties are reported herein. As with true-3-phosphoglyceroyl-enzyme these properties depend on the presence of bound coenzyme (NAD+). The reactivity of the acyl-enzyme toward acceptors (phosphate and arsenate) parallels the extent of its NAD+-induced spectral change [Malhotra, O. P., & Bernhard, S. A. (1973) Proc. Natl. Acad. Sci U.S.A. 70, 2077-2081]. The transcient deacylation of FA-GPDH, preincubated with NAD+, is kinetically biphasic. The relative amplitudes of the fast vs. the slow phase depend on NAD+ concentration but are independent of the nature and concentration of the acyl acceptor. At saturating NAD+ and acceptor concentrations, kinetic biphasicity persists. Perturbation of the acyl-apoenzyme spectrum by NAD+ is also kinetically biphasic. Evidence is presented that the NAD+-requiring acylation of the enzyme results in a protein conformation in which the acyl group is both spectrally perturbed and reactive toward acyl transfer. This acyl-enzyme undergoes a relatively slow isomerization to a conformation in which the acyl spectrum is unperturbed and unreactive in acyl transfer. These two acyl-enzyme conformations are also distinguished by their relative affinities for NAD+; hence, NAD+ is an effector of the conformational equilibrium. Kinetic biphasicity, wherever observed, can be accounted for in terms of two processes: (1) reactivity of the "active" acyl conformation and (2) slow isomerization of the inactive to the active conformation. The two acyl-enzyme conformers are present in finite albeit variable amounts dependent on the extent of NAD+ ligation. Evidence is presented suggesting that each of these conformers has a unique function.

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Spectrophotometric Identification of an Active Site-specific Acyl Glyceraldehyde 3-Phosphate Dehydrogenase

Cited by 151 publications

References 33 publications

Spectral and kinetic studies of imine product formation in the oxidation of p-(N,N-dimethylamino)benzylamine analogs by monoamine oxidase B

Spectral and kinetic studies of imine product formation in the oxidation of p-(N,N-dimethylamino)benzylamine analogs by monoamine oxidase B

Time course of chemical and structural events in protein crystals measured by microspectrophotometry

Role of nicotinamide adenine dinucleotide as an effector in the formation and reactions of acyl-glyceraldehyde-3-phosphate dehydrogenase

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