Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling

Roustan, Claude; Brevet, Annie; Pradel, Louise‐Anne

doi:10.1111/j.1432-1033.1973.tb03135.x

Cited by 29 publications

(5 citation statements)

References 49 publications

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“…Chemical modification studies have indicated that a thiol residue/subunit of creatine kinase can be modified by a variety of other thiol-specific reagents with concomitant loss of activity (Watts et al, 1961;Mahowald et al, 1962;Okabe et al, 1970;Roy et al, 1970;Brown and Cunningham, 1970; Milner-White and Watts, 1971;Roustan et al, 1973). What appears to be an "essential thiol" has been shown in all isoenzymes of creatine kinase and enzymes isolated from various mammalian sources (Watts, 1973), including the one currently under study (Roy et al, 1970).…”

Section: Discussionmentioning

confidence: 99%

Spectrochemical and ligand-binding studies of an active mercurinitrophenol-labeled creatine kinase

Laue¹

1977

Biochemistry

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The formation of several complexes between an active creatine kinase (muscla-type) labeled with 2-mercuri-4-nitrophenol and substrates and anions, singly or in combination. elicited different nitrophenol spectral changes. Blue shifts at different wavelength (between 405 and 426 nm) were observed in all but two spectral changes. Although the Mg2+ cofactor did not produce a change, its presence in the ternary E-MgADP complex caused a perturbation different from that generated by ADP alone. Creatine does not also induce a change. but conferred pronounced effect upon binding to E -MgADP and E-MgADP-nitrate complexes. In a series of complexes that ultimately make up the transition state analogue. E. E -MgADP, E-MgADP-nitrate or E-MgADPcreatine. and E-MgADP-nitrate-creatine, the spectral shifts. both i n the visible and ultraviolet region, were different. These varicd spectral changes and the finding that the rates of reaction of 2 equiv of mercurinitrophenol with the native enzyme in the same series were reduced in the order, 0-, 2-, 3-or 13-, and 100-fold. respectively [Quiocho, F. A,, and Olson, J. 0.A pair of cystcine residues of the muscle-type creatine kinase, or I thiol per subunit, can be reacted with organomercurials such as 2-chloromercuri-4-nitrophenol (Quiocho and Thonison. 1973) or methylmercury chloride (Smith et al., 1975) ibithout the loss of enzymatic activity. The 2-chloromcrcuri-4-nitropheno1, an environment-sensitive spectral probe. can be further utilized to assess the conformational states of creatine kinase in various E-S complexes. For instance. although the stoichiometry of the reaction and enzymatic activity \\ere unaffected. the rates of reaction of 2 equiv of chromophore with a variety of enzyme-substrate complexes were found to be reduced anywhere between 2-to 200-fold relative to the reaction of the native enzyme (Quiocho and Olson, 1974). The postulated transition state analogue EMgADP-nitrate-creatine caused the most severe reduction in rates. Thcse rate differences were attributed to varied changes in the accessibility of the mercury-specific thiol group. I n this communication we wish to report that the spectrum of the nitrophenol incorporated to creatine kinase (without loss ol'activity) can be utilized not only to further assess the complexed states of creatine kinase, but also to monitor structural changes resulting from inactivation by other thiol-specific reagents. We ish also to present evidence, including "peptide mapping" experiment. which indicates that the organomercurial reacts with a thiol group at a site distinct from the active site region. Therefore, the structural change that is characteristic of each enzyme-substrate complex and uniquely ' troni the

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Section: Discussionmentioning

confidence: 99%

Spectrochemical and ligand-binding studies of an active mercurinitrophenol-labeled creatine kinase

Laue¹

1977

Biochemistry

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“…NADP (sodium salt) was provided by Sigma. 2-(Dansy1amino)ethyl monophosphate was synthetized as previously described [19]. Sugars were obtained from Boehringer Mannheim GmbH (Mannheim, Germany), Prolabo or Calbiochem.…”

Section: Methodsmentioning

confidence: 99%

Yeast Hexokinase: Interaction with Substrates and Analogs Studied by Difference Spectrophotometry

et al. 1974

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The mechanism of the reaction catalyzed by yeast hexokinase has been investigated by means of spectrophotometric investigations. Two binding sites, one for the sugar and one for the nucleotide substrate, have been characterized.The interaction of sugar substrates with yeast hexokinase induces spectral perturbations of aromatic residues of the protein molecule. The specificity of the enzyme towards its sugar substrates is discussed.The interaction of ATP-Mg can only be visualized spectrophotometrically when the sugar substrate or inhibitor is already bound to the enzyme.Furthermore, spectrophotometric studies evidence the formation of a binary complex (sugar However, the true mechanism of the reaction and the way in which the ternary complex is formed is still a matter of controversy. properties suggest the formation of a catalytic transitory phosphoryl enzyme [ll]. To explain the glucose-6-phosphate exchange, a second hypothesis in which glucose-6-phosphate is supposed to be bound a t the ATP site and glucose at the sugar site has been postulated [13]. Finally, the binding of some pentoses like lyxose or xylose produces an induced fit of the protein which enhances the hydrolysis rate of ATP [la]. After a n initial activating effect of xylose, De La Fuente has observed that the enzyme is inactivated [15]. I n a recent paper, Colowick has shown that this inactivation is related to the phosphorylation of hexokinase [16]. From this result, he has postulated [ 171 that "catalysis involves an active phosphoryl enzyme derivative which in the presence of D-xylose undergoes rearrangement to form the inactive derivative. It is also possible that the phosphorylation of the protein occurs only as a side reaction".By studying the interaction of various substrates with yeast hexokinase by means of difference spectrophotometry we can provide some information on the mechanism of this reaction; herein, we report the data on the formation of binary and ternary complexes between hexokinase and various substrates and analogs. We have also characterized a binding site specific for nucleotide substrates and another one for sugar phosphorylated sugar.Eur. J. Bioohem. 44 (1974)

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“…From this and the classical studies of Herskowitz [8] we conclude that the formation of the final complex of Scheme 1 results in the removal of one or more tryptophan residues from the interior of the creatine kinase molecule into an exposed polar environment. It is noteworthy that none of the tryptophan residues of the nude enzyme reacts with Koshland's tryptophan reagent [20]. When ethylene glycol is added to the system the amplitude of the difference spectrum decreases but there is a sharp increase as the temperature is decreased.…”

Section: Structural Comments On the Nitrate Complexmentioning

confidence: 99%

Cryoenzymic Studies on the Transition‐State Analog Complex Creatine Kinase ‐ ADPMg ‐ Nitrate ‐ Creatine

Travers

Barman

1980

European Journal of Biochemistry

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Concomitant with the formation of the transition state analog complex creatine kinase . ADPMg . nitrate . creatine [Milner-White, E. J. and Watts, D. C. (1971) Bioclzem. J. 122, 727-7401 there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure.The kinetics of formation and final amplitude of the spectrum were studied at -15 "C using a stopped-flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme-ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine.The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions [Barman, T. E., Brun, A. and Travers, F. (1980) Eur. J. Biochem. 110, 397-4031, This comparison revealed a striking analogy between the two processes. This suggests that the conformation of creatine kinase in the analog complex is very similar to that in the transition state complex on the catalytic pathway.In the preceding paper [I] we used the rapid flowquench apparatus under subzero conditions to search for intermediates in the creatine kinase reaction pathway. Under these conditions the enzyme appeared to be partitioned exclusively between enzyme-substrate and enzyme-product complexes : we could not detect any phosphoenzyme or labile phosphate intermediate down to 4ms. We obtained a complex product (creatine phosphate) progress curve which allowed us to estimate the rate constants pertaining to a simple mechanism. These results make it likely that creatine kinase proceeds via a direct in-line transfer between the participating substrates, as has already been suggested by Milner-White and Watts [2].Another approach towards the study of enzyme mechanisms is to use transition state analogs [3,4]. In the case of creatine kinase there is no simple, single analog inhibitor. However, Milner-White and Watts [2] showed that the combination MgADP, nitrate and creatine is a powerful inhibitor of the enzyme. They suggest that the nitrate binds to the enzyme at the site normally occupied by the transAbbreviations. Cr, creatine; CrP, creatine phosphate. Enzymes. Creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3). ferable phosphate and they conclude that the complex enzyme . ADPMg . NO3 . creatine may mimic the putative transition state complex enzyme . ADPMg . PO4 .creatine. Now, such a complex would occupy a position between an enzyme-substrate and an enzymeproduct complex on the overall catalytic reaction. Since we already have information on the rate constants involved in this step, it seemed to us important to investigate further the formation of the enzyme . ADPMg . NO3 . Cr (nitrate complex) and to see what analogies there might be between the two processes.In this paper we study the formation of the creatine kinase . ADPMg ' NOs ' Cr complex at subzero temperatures [...

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Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling

Cited by 29 publications

References 49 publications

Spectrochemical and ligand-binding studies of an active mercurinitrophenol-labeled creatine kinase

Spectrochemical and ligand-binding studies of an active mercurinitrophenol-labeled creatine kinase

Yeast Hexokinase: Interaction with Substrates and Analogs Studied by Difference Spectrophotometry

Cryoenzymic Studies on the Transition‐State Analog Complex Creatine Kinase ‐ ADPMg ‐ Nitrate ‐ Creatine

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