Preparation and Properties of S‐Cyano Derivatives of Creatine Kinase

Abstract

The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5′‐dithio‐bis(2‐nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol‐blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8‐0.9‐Si‐i group per subunit. This mono‐Scyano derivative of creatine kinase was found 73% as active as the native unniodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted ire the his‐Scyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme.

The effect of limited proteolysis on rabbit muscle creatine kinase

“…If this is so, the integrity of this side chain may well be important in mediating conformational changes associated with the formation of the catalytically active complex. Consistent with this proposal are the observations (i) that small modifications of the cysteine side chain lead to only a small loss of activity, whereas larger modifications lead to greater losses of activity, and (ii) that the extent of inactivation can be roughly correlated with the ability of the modified enzyme to undergo conformational changes associated with the formation of the 'transition-state analogue' complex (Smith et al, 1975;der Terrossian & Kassab, 1976;Keighren & Price, 1978). In the present case it also appears that modification of the environment of the cysteine side chain by proteolysis is associated with an inability of the enzyme to form the 'transition-state analogue' complex.…”

Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid β-protein precursor-like protein-2

“…For this purpose, the modifying reaction has to be highly specific for thiols, it has also to be checked that it is the same thiol residue which is alkylated in each monomer and that no migration of the thiol reagent occurs. Indeed, in some instances, an intramolecular shift of the modifying group from Cys-282 to another thiol of the enzyme has been observed [9][10][11].…”

The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide

“…41000 and each possessing a reactive thiol group (Watts, 1973). It has been known for some time that reaction of this thiol group with a number of reagents, such as iodoacetamide, iodoacetate or Nbs2, leads to inactivation of the enzyme (Watts, 1973), although subsequent work (der Terrossian & Kassab, 1976;Maggio et al, 1977), using modifying reagents that C02 NHCOCH2I…”