Stopped-flow spectrophotometric studies on the reaction of turkey liver xanthine dehydrogenase with reducing substrates.
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ABSTRACT:
SUBMITTER: Fhaolain IN
PROVIDER: S-EPMC1184136 | biostudies-other | 1978 Apr
REPOSITORIES: biostudies-other
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Project description:Studies by e.p.r. (electron-paramagnetic-resonance) spectroscopy and by stopped-flow spectrophotometry on turkey liver xanthine dehydrogenase revealed strong similarities to as well as important differences from the Veillonella alcalescens xanthine dehydrogenase and milk xanthine oxidase. The turkey enzyme is contaminated by up to three non-functional forms, giving molybdenum e.p.r. signals designated Resting I, Resting II and Slow. Slow and to a lesser extent Resting I signals are like those from the Veillonella enzyme, whereas Resting II is very like a resting signal described by K. V. Rajagopolan, P. Handler, G. Palmer & H. Beinert (1968) (J. Biol. Chem. 243, 3784-3796) for aldehyde oxidase. Another non-functional form that gives the Inhibited signal is produced on treatment of the enzyme with formaldehyde. Stopped-flow measurements at 450 nm show that, as for the milk enzyme, reduction by xanthine is rate-limiting in enzyme turnover. The active enzyme gives rise to Very Rapid and Rapid molybdenum(V) e.p.r. signals, as well as to an FADH signal. That these signals are almost indistinguishable from those of the milk enzyme, confirms the similarities between the active sites. There are two types of iron-sulphur centres that give signals like those in the milk enzyme, though with slightly different parameters. Quantitative reduction titration of the functional enzyme with xanthine revealed two important differences between the turkey and the milk enzymes. First, the turkey enzyme FADH/FADH2 system has a redox potential sufficiently low that xanthine is incapable of reducing the flavin completely. This finding presumably explains the very low oxidase activity. Secondly, whereas the Fe/S II chromophore in the milk enzyme has a relatively high redox potential, for the turkey enzyme the value of this potential is lower and similar to that of its Fe/S I chromophore.
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Project description:The binding of the beta-1-4-linked trimer of N-acetyl-D-glucosamine to hen egg-white lysozyme was studied by rapid-reaction-kinetic methods with tryptophyl fluorescence observation of the transients. It was found that discrete segments of the fluorescence-difference spectrum from this reaction were perturbed at different time-points during the binding process. The results were interpretated as the formation of the initial complex, the fast phase of the reaction, perturbing the environment of tryptophan-62 and a subsequent and slower rearrangement of the initial complex perturbing the environment of tryptophan-108. At pH 4.4, the release of protons from aspartate-101 occurred during the rearrangement step of the binding reaction. A model for the reaction is presented (E, enzyme; L, ligand): (see article) The association of this ligand with lysozyme may be visualized in three-dimensional terms as initial complex-formation across the top of the active-site cleft followed by a diving motion of the ligand into the cleft.
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Project description:Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.
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Project description:The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.
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Project description:1. Turkey liver xanthine dehydrogenase engaged in catalysing the oxidation of xanthine by dichlorophenol-indophenol was progressively inactivated by methanol. This inactivation was reversible by NAD(+). 2. Reaction with arsenite and with cyanide, in each case first-order with respect to enzyme, resulted in characteristic alterations in the visible absorption spectrum of the enzyme. The rate of spectral change on reaction with either agent paralleled the rate of loss of enzyme activity. 3. Cyanide inactivation was accompanied by elimination from the enzyme of sulphur as thiocyanate. Partial restoration of activity was effected by incubation with sulphide or with selenide. The results suggest that turkey liver xanthine dehydrogenase, like milk xanthine oxidase (Massey & Edmonson, 1970), contains at the active centre a cyanolysable persulphide group essential to catalytic activity and that selenium may replace sulphur in this group to give an active enzyme. 4. Incubation of the native enzyme with sulphide or with selenide resulted in the rapid loss of half of the xanthine-oxidizing activity, apparently by disrupting the molybdenum and (Fe/S)II loci. This may indicate non-equivalence of the intramolecular electron-transfer systems.
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