New fluorescent peptide substrates for studying the mechanism of gamma-glutamyl hydrolase.

Abstract

gamma-Glutamyl hydroyase (GH), a cysteine protease, catalyzes the hydrolysis of gamma-glutamyl bonds of folates and antifolates and thus plays an important role in the complex regulation of intracellular levels of both these important cofactors and class of drugs, respectively. Improved methods of monitoring GH activity are desired to study its mechanism and kinetics as well as to further our understanding of the specific role of GH in folate and antifolate homeostasis in cells. The slow turnover of substrates containing fluorine adjacent to the scissile bond suggested possible stabilization of an intermediate on the reaction pathway. To facilitate kinetic studies, including the possible identification of a stabilized intermediate on the reaction pathway, a continuous assay for GH based on fluorescence resonance energy transfer (FRET) was developed. Three internally quenched fluorescent peptides, <italic>N</italic>-Me-<italic> p</italic>AB-Glu-gamma-Glu-gamma-Tyr(NO₂), Abz-Glu-gamma-Glu-gamma-Tyr(NO₂), and Abz-(4,4-F₂)Glu-gamma-Glu-gamma-Tyr(NO₂), were synthesized. <italic>N</italic>-Me-<italic>p</italic>AB-Glu-gamma-Glu-gamma-Tyr(NO₂) was shown to provide the first continuous assay for GH that can be used to quantitate GH activity in whole cell lysates. The second fluorescent peptide, Abz-Glu-gamma-Glu-gamma-Tyr(NO₂), provided an improved continuous assay, enabling kinetic constants to be determined of K_m = 5.4 +/- 0.53 muM s<super>-1</super> and k_cat = 0.67 +/- 0.083 s<super>-1</super>. Incorporation of 4,4-difluoroglutamic acid into the third FRET peptide provided steady-state kinetic constants of K_m = 75 +/- 24 muM s<super>-1</super> and k_cat = 0.39 +/- 0.13 s<super>-1</super>. Comparison of the data revealed little change in k_cat but a large increase in K_m, thus indicating that stabilization of an intermediate is not the basis for the observed 20-fold decrease in the rate of hydrolysis of the difluoroglutamate-containing substrate. Transient kinetic studies of the latter two substrates were also carried out. GH-catalyzed hydrolysis of Abz-Glu-gamma-Glu-gamma-Tyr(NO₂) results in a burst, providing the first direct evidence of the formation of an intermediate, presumably the acyl-enzyme, which is consistent with the proposed cysteine protease mechanism for this enzyme.