Modification of the reactive sulfhydryl of bacterial luciferase with spin-labeled maleimides

Abstract

Luciferase from the luminous marine bacterium Beneckea harveyi has been modified at a single cysteinyl residue by reaction with an equimolar amount of a spin-labeled maleimide: 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (I) or 3-(3-maleimidopropyl carbamoyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (II). The resulting enzymes modified with compound I or with compound II were inactive. The apparent second-order rate constants for reaction of luciferase with I and II at pH 7.0, 25 °C, were (5.7 ± 0.1) × 10 4 and (1.1 ± 0.2) × 10 5 m −1 min −1, respectively. Examination of the labeled luciferases by electron spin resonance spectroscopy indicated that the polarity of the region occupied by the nitroxide, and thus presumably of the active center, is similar to that of 2-propanol and that the reactive sulfhydryl resides in a cleft at least 17 Å in length.