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Creatine kinase. Modification of the working enzyme.

ABSTRACT: Protection against inhibition of creatine kinase by iodoacetamide is measured by the decrease in the rate constant for the inhibition reaction; A mixture of purified substrates at equilibrium protects quite strongly when all the components of the mixture are nearly saturating. The protection by substrates 'working' in the forward direction only (from creatine and MgATP) was measured by carrying out the experiment rapidly at low concentrations of the enzyme; by varying the concentration of substrate it was found that the amount of protection when the substrates of the forward reaction are saturating is about 80% (100% protection would imply a value of zero for the rate constant of the inhibition reaction). The effects of Ca2+ and Mg2+ are compared. It is already known that the complex creatine-NO3--MgADP, which is considered to be either a transition-state analogue or an analogue of an intermediate in the reaction pathway, protects fully against iodoacetamide, whereas creatine and MgADP alone, or together without NO3-, do not protect. This suggests that the degree of protection by the working enzyme represents the proportion of enzyme molecules that have a conformation complementary to a creatine-PO3-MgADP intermediate.

SUBMITTER: Milner-White EJ

PROVIDER: S-EPMC1163814 | biostudies-other | 1976 Jul

REPOSITORIES: biostudies-other

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Project description:Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the ?A-crystallin (cryaa) and ?B-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with ?-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and ?-crystallin, we investigated whether ?-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 ?-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between ?-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and ?-crystallin, suggesting that the interaction between CK and ?-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble ?-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and ?-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between ?-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.

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Creatine kinase. Modification of the working enzyme.

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