Alexander Jessica P, Ryan Thomas J, Ballou David P, Coward James K
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Biochemistry. 2008 Jan 29;47(4):1228-39. doi: 10.1021/bi701607v. Epub 2008 Jan 3.
Gamma-glutamyl hydrolase, a cysteine peptidase, catalyzes the hydrolysis of poly-gamma-glutamate derivatives of folate cofactors and many antifolate drugs. We have used internally quenched fluorogenic derivatives of glutamyl-gamma-glutamate and (4,4-difluoro)glutamyl-gamma-glutamate to examine the effect of fluorine substitution adjacent to the scissile isopeptide bond. Using a newly developed continuous fluorescence assay, the hydrolysis of both substrates could be described by Michaelis-Menten kinetics. Fluorine substitution resulted in a significant decrease in observed rates of hydrolysis under steady-state conditions due primarily to a approximately 15-fold increase in Km. Using stopped-flow techniques, hydrolysis of the non-fluorinated isopeptide was characterized by a burst phase followed by a steady-state rate, indicating that formation of the acyl enzyme is not rate-limiting for hydrolysis of this isopeptide. This conclusion was confirmed by analysis of the progress curves over a wide range of substrate concentration, which demonstrated that the acylation rate (k2) is approximately 10-fold higher than the deacylation rate (k3). The increased value of Km associated with the difluoro derivative limited the ability to obtain comparable pre-steady-state kinetics data at saturating concentration of substrate due to inner filter effects. However, even under nonsaturating conditions, a modest burst was observed for the difluoro derivative. These data indicate that either deacylation or rearrangement of the enzyme-product complex is rate-limiting in this isopeptide hydrolysis reaction.
γ-谷氨酰水解酶是一种半胱氨酸肽酶，可催化叶酸辅因子和许多抗叶酸药物的聚γ-谷氨酸衍生物的水解。我们使用了谷氨酰-γ-谷氨酸和（4,4-二氟）谷氨酰-γ-谷氨酸的内淬灭荧光衍生物，以研究与可裂解异肽键相邻的氟取代的影响。使用新开发的连续荧光测定法，两种底物的水解都可以用米氏动力学来描述。氟取代导致在稳态条件下观察到的水解速率显著降低，这主要是由于Km增加了约15倍。使用停流技术，非氟化异肽的水解特征是有一个突发阶段，然后是稳态速率，这表明酰基酶的形成不是该异肽水解的限速步骤。通过分析在广泛底物浓度范围内的进程曲线证实了这一结论，该曲线表明酰化速率（k2）比脱酰化速率（k3）高约10倍。由于内滤效应，与二氟衍生物相关的Km值增加限制了在底物饱和浓度下获得可比的预稳态动力学数据的能力。然而，即使在非饱和条件下，二氟衍生物也观察到适度的突发。这些数据表明，在这种异肽水解反应中，酶-产物复合物的脱酰化或重排是限速步骤。
