A Reaction Mechanism from Steady State Kinetic Studies for 0-Acetylserine Sulfhydrylase from SaLmoneLLa typhimurium LT-2

It has been determined from steady state kinetic studies using the sulfide ion selective electrode that O-acetylserine sulfhydrylase catalyzes a Bi Bi Ping Pong reaction between 0-acetyl-L-serine and sulfide. Both O-acetyl-L-serine (OAS) and sulfide exhibit strong competitive substrate inhibition. A fit of all the data to the equation for the mechanism yields KoAs = 0.149 f 0.059 mM and K,wS = 46% f iO.06 mM for O-acetyl-L-serine and K,z-= 0.066 h 0.004 mM and KISz-= 0.013 i 0.006 mM for sulfide. Product studies varying either substrate at changing fixed levels of cysteine demonstrate that cysteine combines with enzyme at two places along the reaction sequence to produce inhibition with K ,cYs = 1.048 * 0.048 mM and Krc.,, = 11.4 =t 0.5 mM. Relatively high concentrations of acetate are required to produce and at least part of the acetate inhibition due to strength. the of acetate the the and the between and the

The pathway for the biosynthesis of L-cysteine from L-serine, in the enteric bacteria, Escherichia coli and Salmonella typhimurium, was first demonstrated by Kredich and Tomkins (1). They determined that the synthesis proceeded via a two step enzymatic process which may be represented as: It was also observed that there is a physical association of serine transacetylase with 0-acetylserine sulfhydrylase (2), such that a complex could be isolated which catalyzed the synthesis of L-cysteine from L-serine, acetyl-CoA, and H,S. This multienzyme complex, which contained about 5'78 of the total cellular O-acetylserine sulfhydrylase, was given the trivial name cysteine synthetase. The sulfhydrylase activity found in the cell, not associated with serine transacetylase, is identical with that bound to the transacetylase, as far as its physical and kinetic properties are concerned. Michaelis constants obtained for 0-acetylserine sulfhydrylase were 5 x 10m3 M for O-acetyl-L-serine and less than lo-' M for sulfide. 0-Acetylserine sulf'hydrylase is highly specific for O-acetyl-L-serine but both methyl mercaptan and cyanide can replace sulfide as substrate (3,4). The absorption spectrum for the highly purified 0-acetylserine sulfhydrylase (3) exhibits a prominent absorption maximum at 412 nm which is due to the cofactor, pyridoxal phosphate. This maximum is shifted to 470 nm on binding of the substrate 0-acetyl+serine, but in the presence of sulfide, 0-acetyl-L-serine causes no spect?-al shift. Using this spectral shift, Becker et al.
(3) obtained a dissociation constant of 6 x lo-' M, which is 4 orders of magnitude lower than the K,. This large difference was attributed to the fact that the K, is not an accurate indication of substrate binding affinity. 0-Acetylserine sulfhydrylase catalyzes a p substitution reaction, but nothing is known about the order of addition of substrates and release of products. Other PLP' enzymes which 'The abbreviations used are: PLP, pyridoxal phosphate; OAS, O-acetyl-L.-serine; OASS, 0-acetylserine sulfhydrylase; TON, turnover number. 2023 2024 A Reaction Mechanism for 0-Acetylserine Sulfhydrylase catalyze this general type of reaction have been well characterized. Among these is the B protein oftrypotphan synthetase (5) from E. cd, which is responsible for the formation of tryptoph: n from L-serine and indole; and S-methylcysteine or S-ethylcysteine from L-serine and methyl mercaptan or ethyl mercaptan.
The B protein has also been reported (6) to synthesize L-cysteine from L-serine and H,S. This protein has been well characterized by spectral studies (7) Isotope Exchange-The cysteine to sulfide half-reaction was measured in a system containing a reaction vessel (consisting of a 5.0-ml test tube sealed with a serum cap) and a 10% silver nitrate trap (also consisting of a 5.0.ml test tube sealed with a serum cap) which was used to trap sulfide as insoluble silver sulfide. A 1.0.ml volume of the reaction mixture contained the following: 0-acetylserine sulfhydrylase, 0.2 i.u.; [?S]cysteine, 6 mM (0.6 mCi/mmol); sodium sulfide, 0.25 mM; Tris-HCl, pH 7.6, 0.1 M. The control tube contained the same components listed above minus 0-acetylserine sulfhydrylase. After a l-hour incubation period, the reaction was terminated by adding 5 N WC1 and nitrogen was gently bubbled through the mixture for several minutes.
An aliquot from the silver nitrate trap was added to 10 ml of scintillation fluid containing %i volume of Triton X-100, ?/3 volume of toluene, 4.0 g/liter of 2,5-diphenyloxazole (PPO) and 0.2 g/liter of 1,4-bis[2-(4-methyl-5.phenyloxazolyl)]benzene (dimethyl-POPOP Velocity Studies in Absence of Products-When 0-acetyl-L-serine is used as the variable substrate, at fixed varying levels of sulfide, over concentrations of sulfide from 2 x 10m6 M to 3.6 x 10m3 M and of O-acetyl-L-serine concentrations from 2 x low5 M to 5 x lo-' M, an unusual but revealing pattern is obtained (Fig. 4, A and B). The pattern has been shown by Cleland (14) to be indicative of strong competitive inhibition by both substrates in a ping-pong mechanism. When the initial velocity as a function of sulfide as the variable substrate at fixed varying levels of O-acetyl-L-serine is plotted as in Fig. 5, A     Product Inhibition Studies-When 0-acetyl-L-serine was varied at a constant level of sulfide, i.e. one which produced the maximum velocity observed in the sulfide saturation curve (0.25 mM), and changing fixed cysteine, the pattern shown in Fig. 6 was obtained. Since both 0-acetyl-L-serine and cysteine combine to free enzyme, it would be expected that cysteine would be a competitive inhibitor of 0-acetyl-L-serine.
Rut the pattern shown in Fig. 6 indicates a noncompetitive inhibition and the replot of slope uersus cysteine concentration, Fig. 6, is parabolic rather than linear.
Inhibition of the S parabolic, I linear type as found here indicates that the inhibitor combines with the enzyme at two places along the reaction sequence to produce an inhibition. Inhibition constants for cysteine obtained from these data are listed in Table III. With sulfide as the variable substrate, at concentrations which produced low and high levels of inhibition, and a A Reaction Mechanism for 0-Acetylserine Sulfhydrylase 2027  i.e. one which produced the maximum velocity observed in the 0-acetyl-L-serine saturation curve (10 mM), the inhibitory response to cysteine is shown in Fig. 7 (7) and also as cu-aminoacrylic acid in Schiff base with PLP (6), II (Scheme 1). Recently, work by Schnackerz* has shown fairly conclusively, the identity of the species produced on the binding of n-serine to u-serine dehydratase which produces absorption maxima at 460 and 330 nm. The incubation of transition state analogs of II, specifically III, with apodehydratase produced a shift of the absorption maximum of the analog from 420 nm to 460 nm as well as a shoulder at 330 nm. The spectrum of purified 0-acetylserine sulfhydrylase is shown in Fig. 8. Binding of 0-acetyl-L-serine to enzyme (Fig. 9) gives rise, not only to a species at 470 nm, but also to one at 330 nm. When acetate is added to enzyme and 0-acetyl-L-serine ( is attributable to a reversal of the first half-reaction rather than to the high level of ionic strength.
The specificity of acetate in inducing a decreased absorbance at 470 nm was tested with sodium propionate.
The results are shown in Fig. 10 where it may be seen that propionate produces an effect very similar to that caused by acetate. Isotope Exchange--If the reaction mechanism for Oacetylserine sulfhydrylase is of the ping-pong type, then two exchange reactions should be possible since the mechanism requires two half-reactions. These are as follows: where F is a second stable enzyme form, in this case a-aminoacrylic acid in Schiff base with PLP. The 0-acetyl-rJ-serine to acetate exchange was tested using [2-"Clacetate and the cysteine to sulfide exchange was tested using [YI]cysteine.
The results of.these experiments, shown in