EurekaMag.com logo
+ Site Statistics
References:
53,214,146
Abstracts:
29,074,682
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on Google+Follow on Google+
Follow on LinkedInFollow on LinkedIn

+ Translate

Spectrochemical and ligand binding studies of an active mercurinitro phenol labeled creatine kinase






Biochemistry 16(17): 3838-3845

Spectrochemical and ligand binding studies of an active mercurinitro phenol labeled creatine kinase

The formation of several complexes between an active creatine kinase [chicken] (muscle-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 405-426 nm) were observed in all but 2 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 on binding to E-MgADP and E-MgADP-nitrate complexes. In a series of complexes that ultimately make up the transition state analog, E, E-MgADP, E-MgADP-nitrate or E-MgADP-creatine, and E-MgADP-nitrate-creatine, the spectral shifts, both in the visible and UV region, were different. These varied spectral changes and the finding that the rates of reaction of 2 equiv [equivalents] of mercurinitrophenol with the native enzyme in the same series were reduced in the order, 0-, 2-, 3- or 13- and 200-fold, respectively, are manifestations of the various conformational states of creatine kinase. Anion inhibitors-chloride, nitrate or sulfate-caused hypochromic shifts at 408, 406 or 400 nm, respectively, whereas acetate, an activator, brought about a hyperchromic shift at 415 nm. Pyrophosphate inhibitor caused a 439 nm red shift. Further reaction of the nitrophenol-labeled enzyme with mercurinitrophenol, iodoacetamide or 2 equiv of methylmethanethiosulfonate resulted in the modification of another pair of cysteine residues and the loss of activity. The loss of activity occurred concomitant with structural rearrangement that was monitored by the bound chromophore. Further evidence, including the peptide mapping experiment, indicates that the chromophoric probe (at 2 equiv) reacted with 1 crysteine/subunit different from the essential thiol. The structural change that is characteristic of each E-S complex and uniquely monitored by the chromophoric probe presumably occurs elsewhere than at the active site region.

(PDF 0-2 workdays service: $29.90)

Accession: 006460105

PMID: 20128



Related references

Creatine kinase with a thiol bound mercurinitro phenol chromophoric probe. Federation Proceedings 33(5 PART 2): 1234, 1974

Substrate binding to an active creatine kinase ec 2.7.3.2 with a thiol bound mercuri nitro phenol chromophoric probe. Proceedings of the National Academy of Sciences of the United States of America 70(10): 2858-2862, 1973

Nmr studies on nitrogen 15 labeled creatine creatinine phosphocreatine and phosphocreatinine and on barriers to rotation in creatine kinase bound creatine in the enzymatic reaction. Federation Proceedings 45(6): 1751, 1986

Studies on creatine kinase. I. Preparation and amino acid composition of calf brain creatine kinase. II. Hybridation between active and inactive subunits. Biochimie 53(3): 311-320, 1971

Creatine kinase: A role for arginine-95 in creatine binding and active site organization. Biochimica et Biophysica Acta 1546(2): 291-298, 7 April, 2001

Studies on creatine kinase part 1 preparation and amino acid composition of creatine kinase from calf brain part 2 hybridization between active and inactive subunits. Biochimie (Paris) 53(3): 311-320, 1971

Monoclonal antibody studies of creatine kinase antibody binding sites in the amino terminal region of creatine kinase and effects of antibody on enzyme refolding. Biochemical Journal 248(1): 53-60, 1987

Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding. Biochemical Journal 248(1): 53-59, 1987

The stereochemical configuration of manganese ii adp at the active site of creatine kinase elucidated by epr with r p alpha oxygen 17 labeled adp and s p alpha oxygen 17 labeled adp. Journal of Biological Chemistry 257(24): 15047-15053, 1982

Epr and proton relaxation rate studies of spin labeled creatine kinase and its complexes. Journal of Biological Chemistry 246(19): 6029-6036, 1971