Inhibition of adenosine 5′-triphosphate–creatine phosphotransferase by substrate–anion complexes. Evidence for the transition-state organization of the catalytic site

Abstract

1. The substrate combination creatine-MgADP does not significantly protect creatine kinase against inhibition by iodoacetamide in the absence of small anions. 2. Small anions can be divided into three groups according to the way in which they affect creatine kinase: I, acetate reversibly increases enzyme activity in the forward reaction but does not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP; II, planar anions and some halides (HCO(3) (-), HCO(2) (-), NO(3) (-), NO(2) (-), Cl(-), Br(-), F(-)) in the presence of creatine plus MgADP protect the enzyme from inhibition by iodoacetamide; III, tetrahedral anions (SO(4) (2-), HPO(4) (2-), ClO(4) (-), BF(4) (-)) and iodide do not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP but may decrease the protection by class II anions under these conditions. Anions of class II and class III also reversibly inhibit enzyme activity. 3. It is concluded that class II anions form a stable and inactive quaternary enzyme-creatine-MgADP-anion complex and this is responsible for the effect attributed by previous workers to the ternary complex lacking anion. Formation of this complex, particularly in the forward reaction, can lead to markedly non-linear enzyme progress curves. Some previous observations are re-appraised in the light of these findings. 4. From the behaviour of chloride and nitrate ions, and the marked lowering of the K(i) values for creatine and MgADP they produce, it is inferred that planar or monoatomic anions act in the quaternary complex by simulating the transferable phosphoryl group in the transition state (or another intermediate state) of the reaction. 5. It is suggested that, in the course of the reaction, the tetrahedral phosphate-binding site for the transferable phosphoryl group of the substrate (that also binds class II and class III anions) changes into a trigonal bipyramid site (also occupied by class II anions). This strains the phosphoryl group to adopt the transitional sp(3)d hybridized state and must contribute significantly to the low activation energy of the reaction. 6. Catalysis is deduced to proceed by an ;in line' transfer reaction and from the effects of class II anions it is possible to estimate the approximate dimensions of the anionic site in the transition-state complex. 7. The specific protecting effect of an equilibrium mixture of substrates against inhibition by iodoacetamide provides further evidence for the conformational change suggested above as a step in the catalytic process.

Inhibition of myosin ATPase by vanadate ion.

“…Because ADP is required for Vi incorporation, it is further possible that Vi incorporation involves the formation of a binary transition-state analog at the active site (24,29). This type of mechanism has been suggested by Milner-White and Watts (30) to explain the anomalous inhibition of creatine kinase by certain pairs of inhibitors.…”

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases

“…The transition state analog (TSA) complex (Milner-White & Watts, 1971) was formed by dialysis against MgCI2 (5 mM), ADP (4 mM), KNO, (50 mM), and arginine (20 mM), conditions analogous to those that inhibit CK ( Gross et al, 1994). A reducing agent (DTT, 1 mM) and an antibacterial (sodium azide; 0.05%) were added.…”

“…The product was concentrated by ultrafiltration starting with a large volume pressure system, and then down to -I mL by centrifugal filters. The TSA complex (Milner-White & Watts, 1971) was formed by dialysis against 5 mM MgCI2, 4 mM ADP, 50 mM KN03, and 20 mM arginine using the membrane from a 10 kDa centrifugal microconcentrator. Activities were measured by the enzyme linked assay (see above).…”

“…A stable complex can be formed containing creatine (or arginine), Mgt+, ADP, and the nitrate ion, which is thought to mimic the (missing) y-phosphoryl in a planar configuration during transfer (Milner-White & Watts, 1971 ). In the literature, this is referred to as a transition state analog (TSA) complex.…”

Functional non‐identity of creatine kinase subunits of rabbit skeletal muscle

“…CK from rabbit skeletal muscle was purified to homogeneity according to [6]. The enzyme concentration was determined spectrophotometrically, using &SO 7.4x lo4 cm-'.M-' [7]; CK spec. act.…”