Reversible Inactivation and Dissociation of Yeast Hexokinase

Kenkare, Umakant W.; Colowick, Sidney P.

doi:10.1016/s0021-9258(18)96994-2

Cited by 51 publications

(16 citation statements)

References 34 publications

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“…of activity is the fastest, but this may simply reflect an effect of the added substrates which, as detailed above, conspicuously increases the degree of association of the monomers. Kenkare and Colowick (1965) reported that oxidation of SH groups prevented the reassociation of hexokinase, but we were able to exclude this possibility because recovery was unaffected by the addition of 0.04 M ß-mercaptoethanol to the solutions.…”

Section: Results and Their Interpretationmentioning

confidence: 82%

“…A similar stabilization was observed when Na2SG4 was substituted by (NH4)2SG4. Colowick and co-workers (Kenkare & Colowick, 1965; Schulze & Colowick, 1969) concluded from a study of the acid denaturation of hexokinase and the modification of hexokinase by proteases in phosphate buffers that the salts promoted the dissociation of the dimer. We note that this conclusion, and a similar one about glucose, was derived not from direct measurements of the free energy of dissociation but indirectly through the use of kinetic methods, under the assumption that the rate-determining step of the observed reaction was the actual dissociation of the enzyme.…”

Section: Results and Their Interpretationmentioning

confidence: 99%

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Dissociation of yeast hexokinase by hydrostatic pressure

Ruan¹,

Weber²

1988

Biochemistry

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The pressure-induced dissociation of the isozymes P1 and P2 of hexokinase was investigated by studies of the spectral shift of the intrinsic protein fluorescence and by the fluorescence polarization of dansyl conjugates. The free energy of association of the monomers at atmospheric pressure, Katm, was -14.2 kcal mol-1 at 20 degrees C and -11.4 kcal mol-1 at 0 degrees C. The positive enthalpy indicates that the association of the monomers is entropy-driven, overcoming the negative enthalpy of hydration of the subunit interfaces. At 0 degrees C and 1 bar, glucose stabilizes the association by -1.1 kcal mol-1 and the binding of both adenosine 5'-(beta, gamma-methylenetriphosphate) (AMPPCP) and glucose by an even larger amount, -1.34 kcal mol-1. Paradoxically, adenosine 5'-triphosphate (ATP), or AMPPCP, in the absence of glucose destabilizes the association by +0.34 kcal mol-1, while adenosine 5'-diphosphate (ADP) stabilizes it by -0.6 kcal mol-1. Comparison of dV0, the apparent standard volume of association, at different pHs and temperatures indicates that its value (115-160 mL mol-1) is strongly dependent upon the ionization of a group at the subunit interface with a pK near neutrality. Under dissociating pressures, trypsin action results in permanent dissociation of the dimer, confirming earlier observations of Colowick by less direct methods. The P1 and P2 enzymes differ in Katm and dV0 and markedly so in the effects of salt upon the stability of the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Results and Their Interpretationmentioning

confidence: 82%

Section: Results and Their Interpretationmentioning

confidence: 99%

Dissociation of yeast hexokinase by hydrostatic pressure

Ruan¹,

Weber²

1988

Biochemistry

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“…This binding technique appears to be a useful and precise way to determine the purity of hexokinase preparations, although the combining weights given above are only approximate as they are based on the manufacturer's stated concentrations and activities for the enzyme preparations used. The molecular weight of hexokinase has been reported to be 46,000 at pH 7 by Kenkare and Colowick (1965), and to vary from 42,000 to 48,000 by pH 7.6, depending on concentration, by Rudolph and Fromm (1970). It thus appears likely that one CrATP and one glucose combine per molecule of this size.…”

Section: Resultsmentioning

confidence: 99%

Use of chromium-adenosine triphosphate and lyxose to elucidate the kinetic mechanism and coordination state of the nucleotide substrate for yeast hexokinase

Danenberg¹,

Cleland²

1975

Biochemistry

147

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When initial velocities are measured with yeast hexokinase at pH 7, 17 degrees, the inert coordination complex chromium-ATP is competitive vs. MgATP and noncompetitive with glucose, with a dissociation constant of 4-6 muM in either the presence or absence of glucose. These patterns confirm a random kinetic mechanism for this enzyme. With CrATP present, however, the reaction slows down over the first several minutes to a much slower rate, suggesting tighter binding of CrATP with time. When CrATP, MgATP, and D-lyxose are preincubated with the enzyme for 10 min and the reaction started by addition of excess glucose, the dissociation constand of CrATP in now 0.13 muM and the reaction is linear with time. When glucose, CrATP, and enzyme are incubated together and then placed on a Sephadex column, 1 mol each of CrATP and glucose per active center is tightly bound to the enzyme, thus providing a simple and precise method of determining the concentration of active sites. This tight complex, after denaturation with acid, releases 25% free glucose and 75% of a chromium complex containing both ADP and sugar-6-P. CrADP-glucose-6-P is also slowly released from the enzyme during incubation, so that CrATP is actually a very slow substrate. Binding of CrATP with the formation of CrADP-sugar-6-P complexes is also induced by mannose, fructose, glucosamine, 2,5-anhydro-D-glucitol, 2,5-anhydro-D-mannose, and 2,5-anhydro-D-mannitol, while glucose-6-P, 6-deoxyglucose, and lyxose also induce tight binding of CrATP. With excess enzyme, only 25% of CrATP is bound, and the rest does not inhibit the hexokinase reaction. Since bidentate Cr(NH3)4ATP and monodentate CrADP also display inhibition which is tighter with time, but since bidentate CrADP is a poor inhibitor, the actural substrates in the hexokinase reaction appear to be beta, gamma-bidentate MgATP and beta-monodentate MgADP. Tighter inhibition by Cr-8-BrATP than by CrATP suggests that ATP ASSUMES THE SYN CONFORMATION ON THE ENZYME. The substrate inhibition by MgATP induced by the presence of lyxose is shown to be competitive vs. glucose and partial, and, together with other data available, to suggest a kinetic mechanism that is random, but where (1) the rate constant for release of glucose from E-glucose is equal to Vmax, and that for release of glucose from central complexes is less than Vmas; (2) the majority of the reaction flux when both substrates are present at Km levels goes through the path with glucose adding before MgATP, but where at physiological levels the flux through the two paths is more equal. Contd.

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“…The sucrose gradients (5-20 %, w/v) contained 0.05 m Tris-HCl (pH 7.5). The markers used were bovine serum albumin (molecular weight 67,000, j20 = 4.4 S; Hughes and Dintzis, 1964), hexokinase (molecular weight 95,000, s2 = 5.5 S; Kenkare and Colowick, 1965), rabbit muscle glyceraldehyde phosphate dehydrogenase (molecular weight 145,000, s2o = 7.3 S; Allison and Kaplan, 1964), -globulins (molecular weight 160,000, J20 = 7.1 S; Putnam, 1965), and catalase (molecular weight 250,000, s20 = 11.4 S; Martin and Ames, 1961). The sedimentation was followed by elution of the fractions with a special device that included continuous ultraviolet control.…”

Section: Methodsmentioning

confidence: 99%