The binding of coenzymes and analogues of the substrate coenzyme complex to tyrosine aminotransferase

The interaction of tyrosine apo-aminotransferase with coenzymes and coenzyme derivatives has been studied. The derivatives include analogues of substrate-coenzyme complexes formed in the course of the transamination (pyridoxyl derivatives) and coenzyme analogues (pyridoxine 5′-phosphate, 4′-deoxypyridoxine 5′-phosphate, 1-methyl-pyridoxal 5′-phosphate. 1-methyl-pyridoxamine 5′-phosphate). From a comparison of the behaviour of the different compounds, the ΔGo values for the binding of the coenzyme phosphate group and of the substrate (tyrosine) carboxyl and phenyl groups have been determined as equal to -7.1, -3.0 and -2.7 kcal/mol (-29.7, -12.5 and -11.3 kJ/mol) respectively. In the binding of the substrate to the enzyme a significant fraction of the intrinsic ΔGo appears to be used up for some associated endoergonic process. A comparison of the interaction between the enzyme and pyridoxamine 5′-phosphate and some of its derivatives has shown that a positive charge and a large substituent at position 4′ of the coenzyme have an adverse effect on the binding. Methylation of position 1 of pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate makes their ΔGo of binding more positive by 4.0 and 1.8 kcal/mol (16.7 and 7.5 kJ/mol) respectively. ΔHo and ΔSo of binding for the different pyridoxyl-derivatives showed a much greater variability than ΔGo; ΔHo and ΔSo appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme complex at the active site. On the basis of these results a hypothesis on the mode binding of the pyridoxyl-amino acids is presented, which is compatible with the scheme of transamination proposed earlier for aspartate transaminase by Braunstein and by Ivanov and Karpeisky.