The Mechanism of Aldehyde-Induced ATPase Activities of Kinases

22 The Mechanism of Aldehyde-Induced ATPase Activities of Kinases W. W. C L E L A N D and ALAN R. RENDINA

Downloaded by UNIV OF ARIZONA on December 11, 2012 | http://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch022

Department of Biochemistry, University of Wisconsin, Madison, WI 53706

Kinases are enzymes which transfer the γ-phosphate of MgATP to various acceptors. While much is known about the stereochemistry (1), we know little about the chemical mechanism except that the acid-base catalyst which accepts the proton from the alcohol in the hexokinase (2) and fructokinase (3) reactions is a carboxyl group, while that for creatine kinase is a histidine (4). The ATPase activity induced by suitable aldehydes is thus a reaction of considerable interest. Such activity was first seen with glycerokinase (5), which phosphorylates L-glyceraldehyde at the 3 position, but in the presence of D-glyceraldehyde splits MgATP to MgADP and P with no evi dence of any intermediates being formed. It was thought at the time that the ATPase activity involved phosphorylation of the aldehyde hydrate to give the phosphate adduct of the aldehyde, which promptly decomposed. We have now found that this is not correct, and in this paper we will detail studies on 3 enzymes which show ATPase activites in the presence of aldehydes and on two which do not, and discuss the possible chemistry of the reaction. While checking a sample of 2,5-anhydromannose-6-P for fructose-6-P by incubating it with phosphofructokinase and MgATP, we discovered that this aldehyde, which is sterically hindered from forming an internal hemiacetal, induced an ATPase activity (6). Since aldehyde hydration shows a large inverse equilibrium isotope effect of 0.73 when the hydrogen on the carbonyl carbon is replaced by deuterium (7,8) , 2,5-anhydromannose-6-P-1-d will be 60% hydrated, compared to 52% hydration of the unlabeled aldehyde. If the free aldehyde were the activa tor, 48% of the unlabeled and 40% of the deuterated compound would be active, and a normal deuterium isotope effect of i

0.48/0.40 = 1.2 would be seen on V/K (the apparent f i r s t order rate constant) f o r the a c t i v a t o r , while i f the hydrate were the a c t i v e form, an i n v e r s e isotope e f f e c t o f 0.52/0.60 = 0.87 would be seen. The observed value of 1.23 ± 0.03 showed that the free aldehyde and not the hydrate was the a c t i v a t o r (6).

0097-6156/81/0171-0115$05.00/0 © 1981 American Chemical Society

In Phosphorus Chemistry; Quin, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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The absence of an isotope e f f e c t on the V f o r the ATPase r e a c t i o n , however, means that the hydraïe does not bind a p p r e c i a b l y i n the a c t i v e s i t e as a competitive i n h i b i t o r (when a competitive i n h i b i t o r i s present i n the v a r i a b l e s u b s t r a t e , there i s no e f f e c t on V/K, but V i s decreased). A normal isotope e f f e c t on V of 1.2 woïïîi be seen i f the hydrate and f r e e aldehyde had tBe same a f f i n i t y f o r the enzyme. The f a i l u r e of the hydrate to bind presumably r e f l e c t s s t e r i c r e s t r a i n t s i n the a c t i v e s i t e . Since the f r e e aldehyde and a water molecule would take up more space than the hydrate, these data suggest that the aldehyde-induced ATPase does not r e s u l t from i n d u c t i o n by the aldehyde of the proper conformation change to permit r e a c t i o n of MgATP with a bound water molecule. When we attempted to induce ATPase a c t i v i t y by acetate kinase with r e d i s t i l l e d acetaldehyde, a slow r e l e a s e of ADP from MgATP was seen. However, acetaldehyde f r e s h l y passed over a column of Dowex-l-Cl, or condensed from a stream of n i t r o g e n passing over an aqueous s o l u t i o n at n e u t r a l pH d i d not show t h i s a c t i v i t y , and we conclude that the i n i t i a l o b s e r v a t i o n was due to t r a c e s of a c e t i c a c i d . Acetaldehyde d i d c o m p e t i t i v e l y i n h i b i t acetate kinase, however, with a K. of 57 mM f o r unl a b e l e d and 49 mM f o r deuterated acetaldehyde. T h i s i n v e r s e isotope e f f e c t of 0.87 shows that the hydrate i s the i n h i b i t o r y species (acetaldehyde i s 60% hydrated and acetaldehyde-l-d w i l l be 67% hydrated, so that the Κ. of the deuterated species should be lower by the r a t i o 0*60/0.67 = 0.89). Since the a c t i v e s i t e of acetate kinase has room f o r both oxygens of acetate, i t i s not s u r p r i s i n g to f i n d that the hydrate of acetaldehyde i s bound i n the a c t i v e s i t e . The f a i l u r e of the hydrate to be phosphorylated (which would r e s u l t i n ATPase a c t i v i t y ) could be due to i n c o r r e c t geometry, or to the l a c k of an acid-base c a t a l y s t to accept the proton of the hydroxy1 group being phosphorylated. With 3-P-glycerate kinase, which a l s o c a t a l y z e s p h o s p h o r y l a t i o n of a carboxyl group by MgATP, D-glyceraldehyde-3-P d i d not induce ATPase a c t i v i t y . 2,5-Anhydromannose i s phosporylated at the 6-hydroxyl by f r u c t o k i n a s e as w e l l as by hexokinase (9). When we used f r u c t o kinase and hexokinase to check the concentrations of our 2,5anhydromannose p r e p a r a t i o n s (from ADP monitored by a pyruvate kinase, l a c t a t e dehydrogenase couple), more ADP was produced with f r u c t o k i n a s e , and the excess ADP was accompanied by an equal amount of i n o r g a n i c phosphate. 2,5-Anhydromannose i s thus inducing an ATPase a c t i v i t y by f r u c t o k i n a s e as w e l l as becoming phosphorylated. Because of the symmetry of 2,5-anhydromannose, when C-6 i s adsorbed i n the a c t i v e s i t e , phosphory l a t i o n occurs, while when C - l i s adsorbed, MgATP i s s p l i t to MgADP and P.. Since the two r e a c t i o n s are competitive, the product r a t i o equals the r a t i o of V/K values f o r the two react i o n s . Thus: (V/K) /(V/K) = [ADP] / [ A D P ] - 1. With p u r i f i e d 2,5-anhyaromannose the XATPase)/Xkmase) r a t i o X


X

œ



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was 0.13, and was constant from pH 5.5 to 9.4. Since V/K f o r the kinase r e a c t i o n decreases below a pK of 6 (3), the ATPase r e a c t i o n must show the same pH dependence. The (ATPase)/(kinase) r a t i o was higher f o r unlabeled 2,5-anhydromannose than f o r the deuterated compound by a f a c t o r of 1.22, while f o r the two compounds as s u b s t r a t e s f o r f r u c t o kinase (ADP p r o d u c t i o n followed) the V/K isotope e f f e c t was 1.04 ± 0.02, and there was no V isotope e f f e c t . I f only the f r e e aldehyde i s an a c t i v a t o r f o r the ATPase a c t i v i t y , while the f r e e aldehyde and the hydrate have equal V/K values f o r the kinase r e a c t i o n , the p r e d i c t e d isotope e f f e c t on the (ATPase)/ (kinase) r a t i o i s 1.19, while that on the V/K f o r ADP produc t i o n i s 1.024. Thus, the aldehyde i s the a c t i v a t o r f o r f r u c t o kinase as w e l l as f o r phosphofructokinase. With g l y c e r o k i n a s e , the ATPase a c t i v i t y induced by D-glyceraldehyde shows the same pH p r o f i l e f o r the V/K of MgATP as the kinase a c t i v i t y , decreasing at low pH as the group which i s presumably the acid-base c a t a l y s t becomes protonated. Since the most l i k e l y chemical mechanisms f o r the aldehyde-induced ATPase r e a c t i o n s appeared to be metaphosphate cleavage of MgATP or d i r e c t phosphorylation of the aldehyde to give an unstable oxycarbonium i o n , we decided to run the ATPase r e a c t i o n i n the presence of methanol, which should react with metaphosphate to give methyl phosphate, or with a phosphorylated aldehyde to give a phosphoryl methyl a c e t a l . When [ P]-ATP^was incubated with D-glyceraldehyde and g l y c e r o k i n a s e i n 35% [ C]-methanol and the products chromatographed on Doygx-l wi|£ a borate gradient no compounds c o n t a i n i n g both C and Ρ were detected. In a s i m i l a r r e a c t i o n mixture, i n o r g a n i c phosphate and ADP were formed at equal r a t e s , and thus i t appears that i f metaphosphate or a phosphorylated aldehyde were formed, that they reacted with water ( e i t h e r trapped i n the a c t i v e s i t e , or being the f i r s t molecule to have access to the a c t i v e s i t e a f t e r r e a c t i o n ) i n t o t a l preference to methanol. The f a i l u r e to i s o l a t e a s t a b l e methanol and phosphatec o n t a i n i n g compound from ATPase r e a c t i o n s run i n the presence of methanol leaves the chemical mechanism of the ATPase reac t i o n i n doubt. The requirement f o r the proper p r o t o n a t i o n s t a t e of the acid-base c a t a l y s t i m p l i e s e i t h e r that the aldehyde i s phosphorylated to an oxycarbonium i o n , with the negative charge on the acid-base group s t a b i l i z i n g the p o s i t i v e charge on the oxycarbonium i o n , or that the conformation change which produces ATP cleavage r e q u i r e s i o n i z a t i o n of the acid-base catalyst. In the l a t t e r case, ATP could e i t h e r cleave to give metaphosphate or t r a n s f e r a phosphoryl group to a bound water molecule, although the f a i l u r e of the aldehyde hydrates to b i n d makes the b i n d i n g of water i n the a c t i v e s i t e l e s s l i k e l y , and such bound water would be expected to i n t e r f e r e with the normal kinase r e a c t i o n . Perhaps t h i s water i s bound i n such a way that i t has access to the reactants only a f t e r r e a c t i o n , but before they are r e l e a s e d i n t o s o l u t i o n .


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I t i s p o s s i b l e t o t e s t f o r p h o s p h o r y l a t i o n o f the aldehyde by seeing i f 0 i s transferrçg from the aldehyde to phosphate during the ATPase r e a c t i o n . 0 should have a h a l f l i f e i n the carbonyl group o f D-glyceraldehyde i n water a t 20° o f about 50 seconds (10), so by running the r e a c t i o n a t low temperature with a high lçgel o f enzyme and adding the aldehyde i n a small volume o f H^[ 0] i t should be p o s s i b l e t o c a r r y out such an experiment, and we hope to do so i n the near f u t u r e .


Acknowledgement T h i s work was supported by NIH grant GM 18938. Literated Cited

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Knowles, J. R. Annu. Rev. Biochem. 1980, 49, 877. Viola, R. E.; Cleland, W. W. Biochemistry 1978, 17, 4111. Raushel, F. M.; Cleland, W. W. Biochemistry 1977, 16, 2176. 4. Cook, P. F.; Kenyon, G. L.; Cleland, W. W. Biochemistry 1981, 20, 1204. 5. Janson, C. A.; Cleland, W. W. J. Biol. Chem. 1974, 249, 2562. 6. Viola, R. E.; Cleland, W. W. Biochemistry 1980, 19, 1861. 7. Hill, E. A.; Milosevich, S. A. Tetrahedron Lett. 1976, 50, 4553. 8. Lewis, C. A.; Wolfenden, R. Biochemistry 1977, 16, 4886. 9. Raushel, F. M.; Cleland, W. W. J. Biol. Chem. 1973, 248, 8174. 10. Trentham, D. R.; McMurray, C. H.; Pogson, C. I. Biochem. J. 1969, 114, 19. RECEIVED July 12, 1981.


The Mechanism of Aldehyde-Induced ATPase Activities of Kinases

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