The Acetylation of Creatine Phosphokinase with ρ-Nitrophenyl Acetate<sup>*</sup>

Clark, Joan R.; Cunningham, Leon W.

doi:10.1021/bi00888a013

Cited by 13 publications

(5 citation statements)

References 22 publications

(28 reference statements)

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“…Watts (1963) also observed that protection was associated with the increase in pK of the reacting group, inferred to be lysine, by 0.85 of a unit. Although this finding remains valid and is supported by the work of Clark & Cunningham (1965) and Jacobs & Cunningham (1968), the associated conformational change can now be seen to result predominantly from the formation of the enzyme-creatine-MgADP-anion complex; the protection brought about by transphosphorylation in a substrate equilibrium mixture would be minimal under these conditions. Jacobs & Cunningham (1968) also inferred conformational changes to explain their finding that creatine plus MgADP protected creatine kinase against trypsin digestion in excess of either substrate alone.…”

Section: Discussionmentioning

confidence: 72%

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Milner‐White

Watts

1971

Biochemical Journal

196

123

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1. The substrate combination creatine-MgADP does not significantly protect creatine kinase against inhibition by iodoacetamide in the absence of small anions. 2. Small anions can be divided into three groups according to the way in which they affect creatine kinase: I, acetate reversibly increases enzyme activity in the forward reaction but does not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP; II, planar anions and some halides (HCO(3) (-), HCO(2) (-), NO(3) (-), NO(2) (-), Cl(-), Br(-), F(-)) in the presence of creatine plus MgADP protect the enzyme from inhibition by iodoacetamide; III, tetrahedral anions (SO(4) (2-), HPO(4) (2-), ClO(4) (-), BF(4) (-)) and iodide do not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP but may decrease the protection by class II anions under these conditions. Anions of class II and class III also reversibly inhibit enzyme activity. 3. It is concluded that class II anions form a stable and inactive quaternary enzyme-creatine-MgADP-anion complex and this is responsible for the effect attributed by previous workers to the ternary complex lacking anion. Formation of this complex, particularly in the forward reaction, can lead to markedly non-linear enzyme progress curves. Some previous observations are re-appraised in the light of these findings. 4. From the behaviour of chloride and nitrate ions, and the marked lowering of the K(i) values for creatine and MgADP they produce, it is inferred that planar or monoatomic anions act in the quaternary complex by simulating the transferable phosphoryl group in the transition state (or another intermediate state) of the reaction. 5. It is suggested that, in the course of the reaction, the tetrahedral phosphate-binding site for the transferable phosphoryl group of the substrate (that also binds class II and class III anions) changes into a trigonal bipyramid site (also occupied by class II anions). This strains the phosphoryl group to adopt the transitional sp(3)d hybridized state and must contribute significantly to the low activation energy of the reaction. 6. Catalysis is deduced to proceed by an ;in line' transfer reaction and from the effects of class II anions it is possible to estimate the approximate dimensions of the anionic site in the transition-state complex. 7. The specific protecting effect of an equilibrium mixture of substrates against inhibition by iodoacetamide provides further evidence for the conformational change suggested above as a step in the catalytic process.

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Section: Discussionmentioning

confidence: 72%

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Milner‐White

Watts

1971

Biochemical Journal

196

123

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“…Similarly, acetylation of a protein by p-nitrophenylacetate at pH 7 requires a reaction site with properties different from those encountered in normal amino acid side chains (Clark and Cunningham, 1965). Although MgATP2-and MgADPare bound rather strongly by CPK, Kd¡aa = 1 X 10-4 and 6 X 10-5, respectively, at pH 7.9 (Kuby et al, 1962).…”

Section: Discussionmentioning

confidence: 99%

“…A considerable body of evidence (Watts et al, 1961;Kuby et al, 1962;Mahowald et al, 1962;Thomson et al, 1964) suggests that this enzyme has two essentially independent catalytic centers per molecule (mol wt 81,000), and it can be shown that the iodoacetate inhibition results from the conversion of two cysteine residues to S-carboxymethylcysteine (Mahowald et al, 1962;Watts and Rabin, 1962). Inhibition by p-nitrophenylacetate has been less well defined but can be related to the acetylation of two identical but as yet unidentified residues (Clark and Cunningham, 1965) .These reactions appear to be quite independent, since prior inhibition with iodoacetate does not affect the course of subsequent reaction with p-nitrophenylacetate (Clark and Cunningham, 1965). Watts and Rabin (1962) had previously pointed out that the rapidity and independence of pH of the iodoacetate reaction suggests an unusual reactivity of the sulfhydryl group of the protein.…”

Section: Discussionmentioning

confidence: 99%

Cooperative Effects of Substrates and Substrate Analogs on the Conformation of Creatine Phosphokinase^*

Lui¹,

Cunningham²

1966

Biochemistry

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The rate of inhibition of creatine phos-this protection. This observation has allowed a more phokinase by iodoacetamide or p-nitrophenylacetate direct evaluation of previously reported substrateis reduced in the presence of an equilibrium mixture of dependent conformational changes in this enzyme, the substrates of the enzyme: MgATP2-, creatine, Studies of sedimentation constant, reduced viscosity, MgADP-, and phosphocreatine. It has now been shown deuterium exchange, and trypsin susceptibility indicate that the addition of the nonfunctional substrate pair, only small structural alternations in the protein can MgADP-and creatine, is necessary and sufficient for be related to the binding of the combined substrates.Q K_/amuels et al. ( 1961) have reported that creatine phosphokinase (CPK1) undergoes a substrate-dependent change in optical rotation which was interpreted as reflecting a conformational change in the protien. This change was reported to be dependent upon the simultaneous presence of both substrates, and it was suggested that it might play an important role in the mechanism of enzyme action. This hypothesis of a special configuration for "working enzyme" has been employed by Watts and Rabin (1962) to explain the protection against carboxymethylation with iodoacetamide of the two reactive sulfhydryl groups of the enzyme which is afforded by the substrate mixture.

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“…It may be recalled, however, that neither optical rotatory dispersion (Kagi and Li, 1965) nor deuterium-exchange studies (Lui and Cunningham, 1966) could detect conformational change as a result of creatine binding to the MgADP--CrK complex, although this does result in additional protection against trypsin and in the appearance of a marked protection against inhibition by both iodoacetamide and p-nitrophenyl acetate (Lui and Cunningham, 1966). Furthermore, evidence has been presented for the participation of a trypsin susceptible residue, lysine, in the catalytic mechanism of the enzyme (Watts, 1963;Clark and Cunningham, 1965;Jacobs and Cunningham, 1966).…”

Section: Discussionmentioning

confidence: 99%

Creatine kinase. Relations of trypsin susceptibility to substrate binding

Jacobs¹,

Lw²

1968

Biochemistry

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In the presence of Mg2+ and at relatively high enzyme concentrations, creatine kinase is digested by trypsin at pH 9.0 at a slow rate, measureable by pH-Stat analysis. Loss of creatine kinase activity is correlated with the extent of digestion. The rate of trypsin digestion of creatine kinase is decreased in the presence of creatine or adenosine 5 '-diphosphate (ADP) and the effect of this nonworking substrate pair combined is slightly more than additive. On the assumption that the decline in rate of trypsin digestion is a function of substrate binding, dissociation constants have been calculated for creatine and MgADP-(110 (+20) and 0.3 ( i O . 1 ) mM, respectively) and compared with those determined by other methods. In the presence of a saturating concentration of MgADP-, the dissociation C ooperative effects in substrate binding to creatine kinase have been observed in several types of studies of this enzyme and have been ascribed typically to conformational changes resulting from the enzyme-substrate interaction. Lui and Cunningham (1966) have shown that creatine kinase is partially protected by both creatine and MgADP-against digestion by trypsin and that this protection is slightly more than additive. Because only limited insight has been gained from the application of several types of physical methods to the analysis of the postulated conformational change (Samuels, 1961;Lui and Cunningham, 1966;Kagi and Li, 1965), further investigation of the trypsin attack on creatine kinase seemed warranted. These studies have shown that the decrease in rate of trypsin digestion is proportional to the amount of enzyme containing bound substrate. Thus it has been possible to determine dissoci-

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The Acetylation of Creatine Phosphokinase with ρ-Nitrophenyl Acetate^*

Cited by 13 publications

References 22 publications

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Cooperative Effects of Substrates and Substrate Analogs on the Conformation of Creatine Phosphokinase^*

Creatine kinase. Relations of trypsin susceptibility to substrate binding

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The Acetylation of Creatine Phosphokinase with ρ-Nitrophenyl Acetate*

Cited by 13 publications

References 22 publications

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Cooperative Effects of Substrates and Substrate Analogs on the Conformation of Creatine Phosphokinase*

Creatine kinase. Relations of trypsin susceptibility to substrate binding

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Resources

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The Acetylation of Creatine Phosphokinase with ρ-Nitrophenyl Acetate^*

Cooperative Effects of Substrates and Substrate Analogs on the Conformation of Creatine Phosphokinase^*