Abstract
Abstract The spectroscopic measurements of the kinetics and spectra of the enzyme-diphosphopyridine nucleotide compound of glyceraldehyde 3-phosphate dehydrogenase from yeast have been described. A total of 3.5 moles of diphosphopyridine nucleotide are bound to the enzyme (molecular weight, 145,000) as determined by spectrophotometric measurements and direct titration. The ΔE370 on adding diphosphopyridine nucleotide to the enzyme in phosphate buffer is 4.9 cm-1 mm-1. A temperature-dependent enhancement of diphosphopyridine nucleotide binding by phosphate and arsenate was observed. Bicarbonate counteracts the effects of these anions. The inequality of the diphosphopyridine nucleotide binding sites is indicated by the biphasic kinetics for compound formation, differential reactivity to paramercuribenzoate or acetyl phosphate, and selective reactivity in the over-all oxidation reaction. With the use of special spectroscopic techniques it has been possible to measure simultaneously the formation of the enzyme-diphosphopyridine nucleotide compound and the formation of reduced diphosphopyridine nucleotide in the overall oxidation of glyceraldehyde and glyceraldehyde 3-phosphate. These studies indicate that glyceraldehyde 3-phosphate facilitates diphosphopyridine nucleotide binding and that only a small fraction of the total enzyme-diphosphopyridine nucleotide compound could be involved in the oxidation reaction. The major portion of the enzyme-coenzyme complex has kinetic patterns which are incompatible with the possibility that the compound is an active intermediate in the catalytic activity.
Highlights
The molecular extinction coefficient of E .DPN of couraging, but quantitatively unsatisfactory at present for forming any conclusionsthat this complex is a rate-limit.ing or over 4 cm+ rnM+ in the region of 350 rnk is about two-thirds that of DPNH
If the enzyme were treated with charcoalin orderto removethe endogenouDsPN, a ratio of 2.0wasobtained
In our earlierstudies(3,8, 9), the concentrationof the enzyme Wave Length Recording Spectrophotometer-In order to scan wasevaluatedon the basisof a Dzsoof 0.096cm-1mg per ml-l, rapidly through the differencespectrumundervariousconditions, which correspondsto a molecularweight of 120,000(15)
Summary
The spectroscopic measurements of the kinetics and spectra of the enzyme-diphosphopyricline nucleotide compound of glyceraldehyde S-phosphate dehydrogenase from yeast have been described. Presenceof excessglyceraldehydeand3-P-glyceraldehydeshowed The measuremenot f the rate of formation of DPNH in the that “the degree of correlation of the kinetics of the DPN reduction and the enzyme-DPN complex is qualitatively enpresenceof varying concentrations of E.DPN presentsa problem. The molecular extinction coefficient of E .DPN of couraging, but quantitatively unsatisfactory at present for forming any conclusionsthat this complex is a rate-limit.ing or over 4 cm+ rnM+ in the region of 350 rnk is about two-thirds that of DPNH. We have used the double beam spectrophotometerto measuresmall differencesof ab-
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