Mode of Inhibition of P-Hydroxydecanoyl Thioester Dehydrase by 3-Decynoyl-N-acetylcysteamine*

its own destruction by catalyzing the transformation of a substrate analogue to an active site probe of extreme chemical reactivity.

It is concluded that 3decynoyl-NAC as such is not the true inhibitor, but that the dehydrase isomerizes it to the allene which in turn inactivates the enzyme by a chemical reaction. The finding that free 2,3-decadienoic acid inhibits dehydrase, whereas 3-decynoic acid does not, supports the postulated mode of action.
The Esclberichia coli enzyme P-hydroxydecanoyl thioester dehydrase catalyzes the reversible interconversions of P-hydroxydecanoyl, trans.2-decenoyl, and cis-3-decenoyl thioesters (1). Because of its multiple activities dehydrase is considered to be a niultifui~ctional catalyst. The enzyme consists of two identical polgpept,ide chains of 18,000 mol wt, each of which provides a catalytic site (2)

2,S-Decadienoic
Acid-3-Decynoic acid (500 mg) was dissolved in 40 ml of 18% aqueous potassium carbonate and the solution was heated at 90" for 5 hours (5). Crystallization of the crude product removed a large portion of the starting material; the combined mother liquors were concentrated and chromatographed on silicic acid (Unisil, Clarkson Chemical Company) with 5 to 10% ether in pentane.
The rate of IransS-decenoyl-NAC production was measured spectrophotometrically. r---- 2. Effects of deuterium substitution at the a positions of dehydrase inhibitors. Pure acetylenic and allenic thioesters were dissolved in potassium phosphate, pH 6.0, ionic strength = 0.05, and diluted to known concentrations as determined by their ultraviolet spectra.
In each experiment, 33 pg of partially purified dehydras; were added at zero time.
2,2-Dideutero-S-ofecynoyl-NAC-The deuterated acetylenic acid was synthesized by reaction of I-octynyl magnesium bromide with ethylene-d4-oxide (Mallinckrodt/Nuclear, 98% isotopic purity) and oxidation of the resulting alcohol (7) Mass spectral analysis of the thioester confirmed the presence of deuterium in the molecule. Comparison of the deuterated and nondeuterat,ed compounds revealed at least 10 fragments between m/e 81 and 254 differing by 2 mass units.
From these data the isotopic composition was calculated to be 62y0 DD, 30% HD, and 8% HH.
No attempt was made to analyze the absolute deuterium content of the compound. Other Methods-The synthesis of other acyl thioesters has been described (1, 7). Likewise, procedures for enzyme purification, activity measurements, and amino acid analyses have been reported (2, 3, 7). Ultraviolet spectra were recorded with a TJnicam SP-800 dual beam instrument, while fixed wavelength measurements were made with a Gilford 240 single beam spectrophotometer.
A Perkin-Elmer Infracord spectrophotometer was used to analyze infrared absorptions: nuclear magnetic resonances were detected with Varian A-60 or Y-60 instruments. Mass spectra were obtained with an Associated Electrical Industries MS-9 double-focussing instrument.
A4t equal concentrations the allenic isomer diminished enzyme activity at a considerably faster rate than the acetylene (Fig. 1).
Furthermore, inhibition could not be relieved by increasing substrate concentration and therefore appeared to be of the same type as the action of acetylenic thioester on dehydrase. As a more potent enzyme inhibitor than the acetylene, 2,3decadienoyl-NAC may be assumed to react with dehydrase in a more direct fashion.
The question arises whether the allenic and acetylenic inhibitors inactivate dehydrase by different chemical mechanisms or whether the action of the two is re- At various times, 0.5 ml of 3.1 X 10-d M k-3decenoyl-NAC in the same buffer was added and the initial velocity of product formation was recorded. The degree of inactivation was based on comparison with a similar reaction to which no acetylenic thioester had been added.
Since the amount of acetylenic thioester was present at a 60-fold molar excess over dehydrase, the inactivation data were treated in a first order manner. Effects of acetylenic and allenic acids and thioesters on dehydrase activity CH,(CH,),-C = C -CH,CNAC Dehydrase was first incubated in 0.1 M potassium phosphate, pH 7.0, in the presence or absence of acid or thioester inhibitors. The thioester concentration in Experiment 1 was 2 X 10-B M, 41 and the incubation was carried out at 30" for 10 min; in Experiment 2 the acid concentration was 1.6 X low4 M and incubation lasted 30 min.
Aliquots (10 ~1) were subsequently assayed with 4 X lo+ M cis-3-decenoyl-NAC in 0.05 M Tris-HCl, pH 8.0, in a total volume of 1 ml.  A comparison of 3-decynoyl-NAC and 2,2-dideutero+decynoyl-NAC showed a marked difference in the time course of enzyme inhibition ( Fig. 2A). Inactivation by the unlabeled acetylene was significantly more rapid than inactivation by the deuterated acetylene.
When the kinetics of enzyme inactivation by the two compounds were examined, an average value of kH :lin = 2.05 was found (Fig. 3). Correcting this figure for the isotopic composition of the deuterated molecule yielded a value of 2.60 for the ratio of the two rate constants.
These experiments were performed at pH 6.0 in order to minimize nonenzymatic, base-catalyzed acetylene to allene conversion.
Chemical isomerization was, in fact, not measurable at this pH.
The kinetic isotope effect exhibited by the dideuterated acetylene and its absence with deuterated allene implicates removal of an (Y proton as the rate-limiting step in the interaction between enzyme and 3-decynoyl-NAC.
Loss of a proton at the (Y position of the acetylene would, in fact, afford the thermodynamically more stable conjugated allene, and if enzyme-catalyzed, this process would be entirely analogous to olefinic isomerization. The rate differences of acetylene and allene inhibition, now substantiated by the kinetic isotope data, strongly support the notion that enzymatic isomerization is a requisite step in the mechanism by which 3-decynoyl-NAC inactivates dehydrase. It is noteworthy that the magnitude of the above kinetic isotope effect is essentially the same as that observed for removal of a-hydrogen in interactions of substrate with dehydrase. Thus, the observed kH:lcD value for the dehydration of fl-hydroxydecanoyl-NAC to trans-2-decenoyl-NAC was 2.25 (9). Participation of the enzyme in the acetylene-allene conversion would explain why the thioester moiety, a grouping well known to facilitate hydrogen abstraction at a carbons, is essential for the inhibitory action of 3-decynoyl-NAC. The same argument furthermore predicts that the thioester moiety may be less essential or even dispensable for enzyme inhibition by the isomerized product, i.e. once the facilitated removal of a-hydrogen has occurred.
Indeed, 2,3-decadienoic acid, in contrast to 3-decynoic acid, did inhibit dehydrase activity, albeit at very much higher concentrations than the XAC derivative (Table I). Inhibition by the allenic acid or by its NAC derivative could not be reversed by exhaustive dialysis. Free allenic acid inhibits most strongly at pH 5, less so at pH 7, and not noticeably at at pH 9,3 indicating that the protonated species is the active inhibitor.
Assuming an acid dissociation constant of 4.5 to 5, this pH dependence would explain why relatively high concentrations of 2,3-decadienoic acid are needed for inhibition. The action of 2,3-decadienoic acid and its thioester appears to be directed at the same target that is known to be sensit,ive to 3-decynoyl-NAC.
Thus, enzyme exposed to the allenic reagents and subsequently hydrolyzed to its constituent amino acids contained 2 fewer histidyl residues than native dehgdrase. Enzyme treated with 3-decynoyl-NAC showed the same reduction in histidine content (Table II). Of some significance is the observation that 2,3-allenic acids and their derivatives, such as esters or nitriles, are highly susceptible to attack by a variety of nucleophiles (6, 10). In fact, the allenic isomers react much more readily than either 2-or 3-acetylenic derivatives (6). Thus, on the basis of comparative chemical reactivity alone, the allene is clearly the more plausible candidate as the direct enzyme inhibitor. The present experiments would then suggest that the interaction between 3-decynoyl-NAC and dehydrase may proceed as depicted in Fig. 4. The chemical structure of the derivative that is formed when allene reacts with enzyme histidine is currently under investigation.
The present experiments extend our previous understanding of the mode of action of the dehydrase inhibitor 3-decynoyl-XAC. They describe the rather unique case of an enzyme promoting its own destruction by catalyzing the transformation of a substrate analogue to an active site probe of extreme chemical reactivity.