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

L. Li, L. Liu, R. Hong, D. Robertson, J. W. Hastings*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Gonyaulax luciferase is a single-chain (∼137 kDa) polypeptide comprising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each). Each domain has luciferase activity, accounting for the earlier observation that proteolytic fragments (∼35 kDa) of luciferase are active. The activity of the full-length native enzyme is maximal at pH 6.3, dropping to near zero at pH 8; the activity of fragments also peaks at pH 6.3 but remains high at 8. While the activity loss at 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 domains. The three intramolecularly homologous domains, separately cloned and expressed in Escherichia coli as fusion proteins, exhibit pH-activity curves 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 ratio of luciferase activity at pH 8 relative to that at pH 6.3, such that their pH-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 high 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.

Original languageEnglish (US)
Pages (from-to)1844-1849
Number of pages6
JournalBiochemistry
Volume40
Issue number6
DOIs
StatePublished - Feb 13 2001

ASJC Scopus subject areas

  • Biochemistry

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