Creatine kinase active site

Wang S, Wang X, Shi W et al (2008) Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe. Biochim Biophys Acta 1784 415 -22... 

CNDO/2 Theoretical calculations have been used to predict the favoured conformations of creatine and creatine derivatives, including phosphocreatine. These calculations predict that phosphocreatine may adopt conformation (35) to avoid unnecessary steric and electrostatic repulsions.110 The possibility also exists that creatine kinase phosphorylates creatine stereospecifically to form the favoured conformation (35) at the active site of the enzyme. When chicks are fed a diet containing cyclocreatine (l-carboxymethyl-2-iminoimidazole), the phosphorylated... 

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

ATP Triphosphate Chain Conformation. Much of the work in the area of ATP triphosphate chain conformation has been performed by Cleland and co-workers (14--16). Their studies on metal(III)ATP interactions with kinases have led to the classification of kinases according to the stereochemistry of the polyphosphate chain as it binds to the active site. For the kinases they studied (hexokinase, glycerokinase, creatine kinase, phosphofructokinase, 3-phosphoglycerate kinase, acetate kinase, arginine kinase, adenylate kinase and pyruvate kinase) it was found that B, y-bidentate chromi M(III)-ATP (CrATP) and not a,6,y-tridentate CrATP is a... 

In the course of studying the mechanism of action of creatine kinase from rabbit skeletal muscle (M.M isoenzyme), Kenyon and coworkers (4,90) have been involved in the design of specific irreversible inhibitors that are active-site-directed (affinity labels). Creatine kinase catalyzes the reversible transfer of a phosphoryl group ( the elements of "POi") from ATP to creatine, as shown in the following reaction ... 

Additional evidence that epoxycreatine is capable of interaction with the active site of creatine kinase is provided by the observation that epoxycreatine can serve as a substrate in the... 

Affinity labeling ATP sites, 194-96 creatine kinase, 200-205 Amastatin, 94-96 Amide bond hydrolysis, 227 Amino acid sequences, renin inhibitors, 139,141f D-Amlno acids and activity,... 

Such studies on creatine kinase (Eq. 12-31) utilized both a bound Mn2+ ion and a nitroxide spin label to estimate distances of various protons from the nitroxide.683 Together with EPR measurements (Box 8-C), which gave the Mn2+-nitroxide distance, a model of the ATP Mn2 complex in the active site was constructed. Additional EPR experiments on Mn2+ complexes with ATP and ADP containing 170 in the a, (3, or y phospho groups showed that in the enzyme ATP creatine complex the metal ion is bound to all three phospho groups of ATP. It remained coordinated with the two phospho groups of ADP and also that of the phospho-creatine product in the enzyme ADP creatine-P complex as well as in the transition state, which is pictured occurring via a metaphosphate ion.684... 

Figure 12-19 Proposed transition state structure formed from Mn2+i ATP, and creatine bound in the active site of muscle creatine kinase. Based on EPR spectroscopy with regiospecifically 170-labeled substrates. The electrical charges have been added in one possible constellation. However, hydrogen atoms bound to phospho groups are not shown. After Leyh et al.68i...

Creatine kinase is a dimer (molecular weight 82 000), with an active site on each monomer. The activating metal binds to the ATP only, i.e. type I. Spin labelling of the cysteine residue at the active site has allowed distance measurements to Mnu. It appears that Mn remains bound to the a- and /3-phosphoryl groups in ADP. 

Didanosine is a synthetic purine nucleoside analog that inhibits the activity of reverse transcriptase in HIV-1, HIV-2, other retroviruses and zidovudine-resistant strains. A nucleobase carrier helps transport it into the cell where it needs to be phosphorylated by 5 -nucleoiidase and inosine 5 -monophosphate phosphotransferase to didanosine S -monophosphate. Adenylosuccinate synthetase and adenylosuccinate lyase then convert didanosine 5 -monophosphate to dideoxyadenosine S -monophosphate, followed by its conversion to diphosphate by adenylate kinase and phosphoribosyl pyrophosphate synthetase, which is then phosphorylated by creatine kinase and phosphoribosyl pyrophosphate synthetase to dideoxyadenosine S -triphosphate, the active reverse transcriptase inhibitor. Dideoxyadenosine triphosphate inhibits the activity of HIV reverse transcriptase by competing with the natural substrate, deoxyadenosine triphosphate, and its incorporation into viral DNA causes termination of viral DNA chain elongation. It is 10-100-fold less potent than zidovudine in its antiviral activity, but is more active than zidovudine in nondividing and quiescent cells. At clinically relevant doses, it is not toxic to hematopoietic precursor cells or lymphocytes, and the resistance to the drug results from site-directed mutagenesis at codons 65 and 74 of viral reverse transcriptase. 

Creatine kinase was purified from rabbit muscle by the method of Kuby et al, (4). Rabbit muscle pyruvate kinase was purchased from Boehringer. Porcine muscle adenylate kinase was purchased from Sigma, and was further purified by gel filtration on Sephadex G-50. The enzymes were homogeneous as judged by their specific activities and by their migration as single components in sodium dodecyl sulfate gel electrophoresis. Proton NMR spectra at 250 MHz of 0.5-2.0 mM enzyme sites in 0 solution were obtained with a Bruker WM 250 MHz pulse FT spectrometer at 25°. At least 256 transients were accumulated over 8192 data points using 16 bit A/D conversion. Relaxation rates and histidine pK values were determined by standard NMR methods (5, 6),... 

Figure I. Composite diagram summarizing the roles of histidine imidazole groups at the active sites of the phosphotransferase enzymes creatine kinase (CrK), pyruvate kinase (PK), and adenylate...

An example of a random type of reaction is creatine + ATP creatine phosphate + ADP, which is catalyzed by creatine kinase (see Chapter 20). In this case, creatine and ATP are bound to the active site in either sequence, and after the transfer of the phosphate group of the bound ATP to the bound creatine both products are released in either sequence. 

Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188-190, 1967 Chen, R.F., Huorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride. Arch. Biochem. Biophys. 128, 163-175, 1968 Chen, R.F., Dansyl-labeled protein modified with dansyl chloride activity effects and fluorescence properties. Anal Biochem. 25, 412M16, 1968 Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride. Biochemistry 9, 3878-3885, 1970 Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride activity effects and fluorescence properties. 

S. Reddy, A. Jones, C. Cross, P. Wong, A. Van Der Vliet, Inactivation on Creatine Kinase by S-Glutathionylation of the Active-Site Cysteine Residue, Biochem 7347 (2000) 821-827. 

Reddy S, Jones AD, Cross CE, Wong PS, Van Der Vliet A (2000) Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue. Biochem J 347 821-7 Holmgren A (1990) Glutaredoxin structure and function. In Vina J (ed) Glutathione metabolism and physiological functions. CRC, Boca Raton, Florida, pp 145-15430 Holmgren A (1984) Enzymatic reduction-oxidation of protein disulfides by thiore-doxin. Methods Enzymol 107 295-300... 

Therefore, the complex bound at the active site must be the A isomer shown above. Similarly, (5p)-[y3- 0]ATP complexed with Mn(ll) at the active site of creatine kinase with an unreactive substrate analog elicits the effect, whereas ( p)-[/3- 0]ATP does not. Therefore, the coordination stereochemistry of MnATP at the active site of creatine kinase is fully defined as that shown below (25). 

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