Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.     

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.     

Studies on the sucrose phosphate synthetase kinetic mechanism

The kinetic properties of wheat germ sucrose phosphate synthetase, which catalyzes the reaction UDP-glucose + fructose 6-phosphate → UDP + sucrose 6-phosphate have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. A dead-end inhibitor, inorganic phosphate, was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. The results of initial velocity and product and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

The kinetic mechanism of the anterior pituitary progesterone 5 alpha-reductase

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.     

Autoacetylation of the histone acetyltransferase Rtt109

Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear. Here, we examined the mechanism of Lys-290 acetylation and found that Rtt109 catalyzes intramolecular autoacetylation of Lys-290 ～200-times slower than H3 acetylation. Deacetylated Rtt109 was prepared by reacting with a sirtuin protein deacetylase, producing an enzyme with negligible HAT activity. Autoacetylation of Rtt109 restored full HAT activity, indicating that autoacetylation is necessary for HAT activity and is a fully reversible process. To dissect the mechanism of activation, biochemical, and kinetic analyses were performed with Lys-290 variants of the Rtt109-Vps75 complex. We found that autoacetylation of Lys-290 increases the binding affinity for acetyl-CoA and enhances the rate of acetyl-transfer onto histone substrates. This study represents the first detailed investigation of a HAT enzyme regulated by single-site intramolecular autoacetylation.     

Structure and histone binding properties of the Vps75-Rtt109 chaperone-lysine acetyltransferase complex

The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4)(2) tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4)(2). Investigation of Vps75-Rtt109-(H3-H4)(2) and Vps75-(H3-H4)(2) complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.