An essential histidine ligand to the electron transfer copper (Cu H) of peptidylglycine α-hydroxylating monooxygenase (PHMcc) was mutated to an alanine and found to retain copper binding and hydroxylase activity [Jaron, S., et al. (2002) Biochemistry 41, 13274-13282]. An extensive kinetic and deuterium isotope effect study finds this mutant to maintain full coupling of O2 consumed to product formed despite a 3 order-of-magnitude decrease in kcat and a 300-fold decrease in k cat/Km(O2). Unexpectedly, electron transfer is not rate-limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on kcat of 3.27 ± 0.39 suggests that C-H bond cleavage has become more rate-limiting, implicating a role for His172 that goes beyond that of a simple ligand to CUH. The mechanistic implications are discussed.
|Original language||English (US)|
|Number of pages||11|
|State||Published - Dec 26 2006|
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