N-terminal intramolecularly conserved histidines of three domains in Gonylaulax luciferase are responsible for loss of activity in the alkaline region

Gonyaulax luciferase is a single-chain (similar to 137 kDa) polypeptide com prising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each). Each domain has luciferase activity, accoun ting for the earlier observation that proteolytic fragments (similar to 35 kDa) of luciferase are active. The activity of the full-length native enzym e is maximal at pH 6.3, dropping to near zero at pH 8; the activity of frag ments also peaks at pH 6.3 but remains high at 8. While the activity loss a t higher pH might be thought to be associated with the conformation of the full-length protein, we show here that this is a property of individual dom ains. The three intramolecularly homologous domains, separately cloned and expressed in Escherichia call as fusion proteins, exhibit pH-activity curve s similar to that of the full-length enzyme. For each domain the removal of approximately 50 N-terminal amino acids resulted in an increase in the rat io of luciferase activity at pH 8 relative to that at pH 6.3, such that the ir pa-activity profiles mimicked that of the proteolytic fragments reported earlier. Replacement of N-terminal histidines by alanine by site-directed mutagenesis identified four that are involved in the loss of activity at hi gh pH. This system illustrates an unusual, possibly unique mechanism for pH regulation of enzyme activity, which has been postulated to be responsible for the control of the characteristic flashes of bioluminescence.