PECULIAR SPECTROSCOPIC AND KINETIC-PROPERTIES OF CYS-47 IN HUMAN PLACENTAL GLUTATHIONE TRANSFERASE - EVIDENCE FOR AN ATYPICAL THIOLATE ION-PAIR NEAR THE ACTIVE-SITE

Cys-47, the most reactive cysteine in the homodimeric glutathione tran sferase (EC 2.5.1.18) from human placenta (class Pi), displays peculia r acid base and spectroscopic properties. The thiolate form of this re sidue is characterized by a sharp UV absorption spectrum centered at 2 29 nm with an epsilon = 7,500 M-1 cm-1. The dependence of the apparent extinction coefficient on pH indicates that the sulfhydryl group of C ys-47 has a pK(a) value of 4.2. Moreover the dependence of the reactiv ity of Cys-47 toward bromopyruvate and iodoacetamide with pH resembles that found for the functional sulfhydryls of thiol proteases, which h ave very low pK(a) values and exist mainly as a mercaptide-imidazole i on pair. The apparent pK(a) value for Cys-47, calculated by this kinet ic approach, is in good agreement with that determined spectroscopical ly. X-ray crystallographic data indicate that the protonated amino gro up of Lys-54, 4.9 angstrom from the sulfur atom, is probably involved in the deprotonation of Cys-47. Calculation of the electrostatic poten tial on the sulfur atom of Cys-47 gives a theoretical pK(a) value of 3 .5 for the sulfhydryl group. The simulated neutralization of Lys-54 sh ifts the pK(a) value of Cys-47 to a normal value of 9.5. These finding s suggest that at physiological pH values, Cys-47 exists as the thiola te ion stabilized by an ion pair formation with the protonated amino g roup of Lys-54, and this probably accounts for its high reactivity.