The use of chromium(III) and cobalt(III) complexes of adenosine diphosphate to elucidate the catalytic mechanisms of creatine kinase

Abstract

Complexes of the type α,β-bidentate Cr(H 2O) 4-n− (NH 3 nADP (for n = 0, 3, 4) and Co(H 2O) 4-m− (NH 3 m (for m = 3, 4) have been used over the pH range 5.5 to 7.8 to probe the chemical mechanism of rabbit muscle creatine kinase. In addition, V, V/K and Km values have been obtained for the normal substrate, MgADP, over the same pH range. The cobalt and chromium nucleotides were synthesized following the procedures given in ref. 1–5. Separation of the diasteromers into Λ and Δ screw sense isomers was by cycloheptaamylose chromatography (pH 5.5, 4°C, 10 m M MES, 2.5 cm × 2 m). The first band of the column (Δ isomer) was used in all inhibition studies. Kinetic assays with creatine kinase used the hexokinase-glucose-6-phosphate dehydrogenase coupled assay in which the appearance of NADPH at 340 nm was monitored. The pH was maintained with 50 m M MES buffer between pH 5.5–6.8 while 50mM HEPES was used from pH 7.0–7.8. No buffer effects were observed. All of the cobalt and chromium nucleotides acted as competitive inhibitors versus MgADP and were fit to the equation v = VS/{K(1 + I/Kis) + S}. The Kis and Km values for the metal nucleotides as a function of pH are shown in Fig. 1. The data indicated that metal-nucleotide binding to the enzyme is strongest below an approximate pK of 6.4. This pK is not associated with the metal nucleotide complexes nor a binding group of the enzyme. It appears that the pK of the acid-base catalyst (thought to be histidine [6] is about 6.4 in the absence of nucleotide, and is raised to about 7.2 in ▪ the presence of a nucleotide, presumably as a result of a protein conformation change which allows a hydrogen bond to form between the histidine and the phosphorylated nitrogen of creatine phosphate. This change in pK upon nucleotide binding necessarily causes tighter nucleotide binding at low pH. Additionally, it is observed that chromium nucleotide are bound more strongly than the analogous cobalt nucleotide and that the binding affinity decreases as ammonia ligands are substituted for water in the metal coordination sphere.