The effect of substrate concentration and pH on the enzymic sulphation of L-tyrosyl derivatives.

1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to derivatives of l-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH7.5 and 37 degrees C the K(m) values for l-tyrosine methyl ester and adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate are 0.3mm and 8nm respectively. The K(m) value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the K(m) and V(max.) values for l-tyrosine methyl ester, tyramine and N-acetyl-l-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH7.5 and 30 degrees C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate but not by l-tyrosine methyl ester.