高粱叶片PEP羧化酶的溶液构象——近紫外圆二色性光谱研究

用近紫外CD光谱技术追踪了PEP羧化酶与各种配基的相互作用。底物PEP、必需金属离子Mg~( )、PEP-Mg~( )以及效应剂G6P、Gly、G6P-Gly,均可引起高粱叶片PEP羧化酶近紫外CD光谱各不相同的变化。这表明高粱叶片PEP羧化酶分子构象有较大的灵活性,不同的配基与酶相互作用可引起酶分子不同的构象变化,因而使酶分子表现出催化功能、调节特性、必需氨基酸残基的化学反应性以及稳定性诸方面的差异。

NEAR UITRAVIOLET CIRCULAR DICHROISM STUDIES ON THE CONFORMATION OF SORGHUM LEAF PHOSPHOENOLPYRUVATE CARBOXYLASE IN SOLUTION

The near ultraviolet CD spectra of PEP carboxylase from sorghum leaves were studied in the presence of various ligands, The free enzyme shows a spectrum with a negative CD peak at 275 nm and two shoulder at 268nm and 295nm. The spectrum of enzyme-PEP complex indicates Changes in the asymmetric microenvironments of Phe and Tyr residues. Mg~( ) binding to the enzyme giyes rise to changes in the CD absorption of Tyr residues in the enzyme moleeuies (see Fig. 1). Combined addition of PEP and Mg~( ) to the enzyme solution produces profound effect on the enzyme CD spectrum. Compared with the CD spectrum of free enzyme, the presence of PEP-Mg~( ) complex causes a blue shift in the negative CD peak (from 275nm to 272nm) and appearance of a new shoulder at 285nm (see Fig.2).Effectors G6P and Gly cause very different changes in the spectrum of the enzyme (see Fig.3). Gly exerts effect on the enzyme CD spectrum mainly in the region of Tyr absorption, while the binding of G6P to the enzyme gives rise to vigorous changes in the microenvironments of the three aromatic residues.All these results proved that the molecule of PEP carboxylase is highly flexible and that multiple conformational states of the enzyme can be induced by interaction with substrate, Mg~( ), effectors, etc. The flexibility could explain the obvious. differences in catalytic activity, regulatory properties and stability of the enzyme in the presence of various ligands.