Czerwinski R M, Johnson W H, Whitman C P
Medicinal Chemistry Division, College of Pharmacy, University of Texas, Austin 78712-1074, USA.
Biochemistry. 1997 Nov 25;36(47):14551-60. doi: 10.1021/bi971545h.
The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate tautomerase has been mutated to Gly, Ala, Val, and Leu, residues with aliphatic side chains. The Val mutant was partially (55%) processed by removal of the amino-terminal methionine to yield P1V/M1P2V, while the Leu mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in kcat with no change in Km, and the residual activity of the unresolvable P1V/M1P2V mixture could be explained by the summation of two activities, one equal to that of M1P2L and the other equal to that of the P1G mutant. The P1G and P1A mutants showed 76- and 58-fold decreases in kcat and much smaller decreases in Km of 4- and 2.8-fold, respectively. The dissociation constant of the substrate analog cis,cis-muconate decreased 1.7-fold in the P1G mutant as determined by NMR titration. 2D 1H-15N HSQC spectra and 3D 1H-15N NOESY HSQC spectra of the 15N-labeled P1G mutant showed no structural differences from the wild-type enzyme except for small changes in backbone 15N and NH chemical shifts at the active site. Both the P1G and P1A mutants showed no change in overall conformation by circular dichroic spectroscopy. Both mutants and the wild-type enzyme generate the S-enantiomer of the product [5-2H]-2-oxo-3-hexenedioate with comparable stereoselectivities indicating a largely intact active site. The P1G and P1A mutants showed 10- and 4-fold decreases, respectively, in catalysis of exchange of the C3 proton of the substrate 2-oxo-1,6-hexanedioate, consistent with the lower basicities of Gly-1 and Ala-1 compared to Pro-1. The pH dependences of kcat/Km for the P1G and P1A mutants revealed pKa values of the general base of 5.3 and 5.9, respectively. NMR titration of the uniformly 15N-labeled P1G mutant showed the pKa of Gly-1 to be < or = 5.6, in agreement with the kinetic data. As with the wild-type enzyme, the active site environments on the P1G and P1A mutants lower the pKa of the general base by at least 2.5 units. It is concluded that the 2 order of magnitude decreases in kcat in the P1G and P1A mutants result from both a decrease in basicity and an increase in flexibility of the general base. The greater 10(3.4)-fold decrease in kcat found with the presence of an additional residue at the amino-terminus is ascribed to either the complete blockage or the drastically altered position of the general base.
4-草酰巴豆酸互变异构酶的催化性通用碱Pro-1已突变为甘氨酸、丙氨酸、缬氨酸和亮氨酸，这些残基带有脂肪族侧链。缬氨酸突变体通过去除氨基末端甲硫氨酸进行了部分（55%）加工，产生P1V/M1P2V，而亮氨酸突变体未加工，完全保留了甲硫氨酸（M1P2L）。M1P2L突变体的催化常数（kcat）降低了2300倍，米氏常数（Km）无变化，无法拆分的P1V/M1P2V混合物的残余活性可以通过两种活性的总和来解释，一种与M1P2L的活性相等，另一种与P1G突变体的活性相等。P1G和P1A突变体的kcat分别降低了76倍和58倍，Km的降低幅度小得多，分别为4倍和2.8倍。通过核磁共振滴定法测定，底物类似物顺，顺-粘康酸的解离常数在P1G突变体中降低了1.7倍。15N标记的P1G突变体的二维1H-15N HSQC谱和三维1H-15N NOESY HSQC谱显示，除了活性位点处主链15N和NH化学位移有小的变化外，与野生型酶没有结构差异。通过圆二色光谱法，P1G和P1A突变体的整体构象均无变化。两种突变体和野生型酶生成产物[5-2H]-2-氧代-3-己烯二酸的S-对映体，具有相当的立体选择性，表明活性位点基本完整。P1G和P1A突变体在催化底物2-氧代-1,6-己二酸的C3质子交换时分别降低了10倍和4倍，这与甘氨酸-1和丙氨酸-1相对于Pro-1较低的碱性一致。P1G和P1A突变体的kcat/Km对pH的依赖性分别显示通用碱的pKa值为5.3和5.9。对均匀15N标记的P1G突变体进行核磁共振滴定显示，甘氨酸-1的pKa≤5.6，与动力学数据一致。与野生型酶一样，P1G和P1A突变体上的活性位点环境使通用碱的pKa至少降低2.5个单位。得出的结论是，P1G和P1A突变体中kcat降低两个数量级是由于通用碱的碱性降低和柔性增加。在氨基末端存在一个额外残基时发现kcat降低幅度更大（10(3.4)倍），这归因于通用碱的完全阻断或位置的剧烈改变。
