The resonance-stabilized quinonoid 5-mercapto-2-nitrobenzoate (TNB) is a substrate for O-acetylserine sulfhydrylase-A (OASS-A) and -B (OASS-B), giving rise to the product S-(3-carboxy-4-nitrophenyl)-L-cysteine (S-CNP-cysteine) as confirmed by ultraviolet-visible and 1H NMR spectroscopies. A comparison of the kinetics of OASS-A and OASS-B indicates that the mechanism proceeds predominantly via a bi-bi ping pong kinetic mechanism as suggested by an initial velocity pattern consisting of parallel lines at low concentrations of reactants, but competitive inhibition by both substrates as the reactant concentrations are increased. Thus, in the first half-reaction, O-acetyl-L-serine (OAS) or beta-chloro-L-alanine (BCA) is converted to alpha-aminoacrylate in Schiff base with the active site pyridoxal 5'-phosphate, while in the second half-reaction cysteine (with sulfide as the reactant) or S-CNP-cysteine (with TNB as the reactant) is formed. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive. These data are consistent with acetate reversing the first half-reaction and producing more free enzyme to which acetate may also bind. Thus, there may be some randomness to the mechanism at high concentrations of the nucleophilic substrate.

Protein glycation by hexoses has been implicated in the pathophysiology of a number of diseases as well as the aging process. Studies of ADP-ribose polymer metabolism have shown that free ADP-ribose is generated at high rates in the cell nucleus following DNA damage. Protein glycation by ADP-ribose has been reported although the chemistry is not understood. Described here is the synthesis and characterization of model conjugates for protein glycation of lysine residues by ADP-ribose. Two stable conjugates derived from ADP-ribose and n-butylamine were isolated and characterized. Both conjugates were shown to be ketoamines derived from a Schiff base by an Amadori rearrangement. The chemical stability of the ketamines allowed them to be differentiated from all classes of enzymic protein modification by ADP-ribose. Further, their chemical properties suggest that a previous report of histone H1 modification in carcinogen treated cells was due to glycation by ADP-ribose.

As substrate for protein-mono-ADP-ribosyltransferases, NAD has been shown to be the donor of ADP-ribose to many different nucleophiles found in proteins. This post-translational modification of proteins has been implicated in the regulation of membrane-associated processes including signal transduction, muscle cell differentiation, and protein trafficking and secretion. Described here is the preparation and chemical characterization of low molecular weight conjugates that were used as models for an acetal linkage between ADP-ribose and the hydroxyl group of a protein acceptor such as serine, threonine, tyrosine, hydroxyproline, or hydroxylysine residues. Model conjugates of ADP-ribose containing an acetal linkage were prepared, their structures were established by NMR, and the chemical stability of the linkage to ADP-ribose was studied and compared to the other known ADP-ribosyl-amino acid linkages. The rapid release of intact ADP-ribose from the acetal model conjugates in 44% formic acid distinguished them chemically from all the other known ADP-ribosyl-amino acid modifications. Rat liver proteins were shown to be modified by ADP-ribose in vivo by acid-labile linkages, providing evidence for a new class of endogenous ADP-ribose modification of animal cell proteins. The amount of modification was approximately 16 pmol of ADP-ribose per mg of total protein, and proteins modified by acid-labile linkages were detected in all subcellular fractions examined, suggesting that the scope of this modification in vivo is broad.

Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies. Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate. The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively. Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0. D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH. The alpha-secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single-wavelength stopped-flow experiments. Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction.