Mechanistic studies on the alkyltransferase activity of serotonin N-acetyltransferase

Background: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransfera se, AANAT) catalyzes the first, rate-limiting step in the biosynthesis of t he circadian hormone melatonin (5-methoxy-N-acetyltryptamine) from scrotoni n. Our recent discovery that, in addition to catalyzing the acetyl transfer from acetyl-coenzyme A (acetyl-CoASH) to serotonin, AANAT is also a robust catalyst for the alkyl transfer reaction between CoASH and N-bromoacetyltr yptamine has not only opened up a new way to develop cell-permeable AANAT a cetyltransferase inhibitors that are valuable in vivo tools in helping eluc idate melatonin's (patho)physiological roles, but has also raised a questio n - how does AANAT accelerate the alkyl transfer reaction? In this study, m echanistic aspects of the AANAT-catalyzed alkyl transfer reaction were expl ored by employing CoASH and a series of N-haloacetyltryptamines that were a lso evaluated for their AANAT acetyltransferase inhibitory activities. Results: Investigation of various N-haloacetyltryptamine analogs showed a s imilar leaving group effect on the enzymatic and non-enzymatic reaction rat es. Steady-state kinetic analyses demonstrated that AANAT alkyltransferase obeys a sequential, ternary complex mechanism, with random substrate bindin g. Rate versus pH profiles revealed the catalytic importance of an ionizabl e group with pK(a) similar to 7. All those N-haloacetyltryptamines that ser ve as substrates of AANAT alkyltransferase are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. In particula r, N-chloroacetyltryptamine was also shown to be a potent inhibitor of intr acellular melatonin production in a pineal cell culture assay. Conclusions: This is the first detailed investigation of the alkyltransfera se activity associated with an acetyltransferase. Our results indicate that AANAT does not accelerate the alkyl transfer reaction by simple approximat ion effect as previously proposed for the similar alkyl transfer reaction c atalyzed by other acyltransferases. This study has general implications for developing novel inhibitors by taking advantage of the promiscuous alkyltr ansferase activity associated with several acyltransferases. (C) 2001 Elsev ier Science Ltd. All rights reserved.