Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.

Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein (SLRP) family that was initially identified in the eye in association with the collagen fibrils of the vitreous humor. Recombinant and tissue-extracted forms of bovine opticin were subjected to biochemical and biophysical characterization. Following SDS-PAGE the predominant component produced by both forms was a broad band between 45-52 kDa. There was evidence for two-stage processing and, additionally, a proteolytic cleavage product of ϳ25 kDa. Deconvolution of circular dichroism spectra revealed ␤-sheet (41%), ␤-turn (21%), and ␣-helix (10%), and thermal denaturation experiments showed a transition with a midpoint of 47°C. Weight-averaged molecular mass measurements using both light scattering and analytical ultracentrifugation demonstrated that opticin exists in solution as a stable dimer of ϳ90 kDa, which can be dissociated into a monomer by denaturation with 2.5 M guanidine hydrochloride or during SDS-polyacrylamide electrophoresis. Opticin remains a dimer after removal of the amino-terminal region by O-sialoglycoprotein endopeptidase digestion, suggesting that dimer formation is mediated by the leucine-rich repeats. Dimerization could have a number of functional consequences, including divalent ligand interactions.The extracellular matrix small leucine-rich repeat proteins (SLRPs) 1 are a family of molecules to which various functions have been ascribed, including regulation of matrix assembly, binding to growth factors, and suppression of cell growth (for review, see Ref. 1). They contain a varying number of leucinerich repeats (LRRs) that contain the consensus sequence LXX-LXLXXNXL, where X is any amino acid, L is leucine, isoleucine or valine, and N can be asparagine, cysteine or threonine. The LRRs are generally flanked by 4 cysteine residues at the amino-terminal end and two at the carboxyl-terminal end. The SLRPs have been subdivided into three classes depending upon the number of LRRs, the spacing of the four amino-terminal cysteine residues, and the number of exons encoding the gene (1, 2). The class I and II SLRPs possess ϳ12 LRRs, whereas the class III members, including opticin (3), epiphycan/PG-Lb (4, 5) and osteoglycin/mimecan (6, 7), have ϳ7 LRRs.Opticin was first identified in a 4 M guanidine hydrochloride (GdnHCl) extract of collagen fibrils from the vitreous humor of the eye (3). Opticin expression is largely confined to the eye and, in the mouse, was localized specifically to the non-pigmented epithelium of the ciliary body (8). However, expressed sequence tag analyses of adult human iris and retinal pigment epithelium/choroid (available at neibank.nei.nih.gov/index. shtml) suggest that opticin is a very common transcript in these tissues (9 -11). Furthermore, recently published immunolocalization data suggests the presence of opticin in a number of ocular tissues, including the cornea, iris, ciliary body, vitreous, choroid, and retina (12). Friedman et al. (12) also suggest that mutations in th...

Although SAT (serine acetyltransferase) of Escherichia coli, which catalyses the first step in cysteine synthesis, proceeds via a random-order ternary complex reaction mechanism [Hindson and Shaw (2003) Biochemistry 42, 3113-3119], it has been suggested that the nearly identical enzyme from Salmonella typhimurium might involve an acetyl-enzyme intermediate [Leu and Cook (1994) Protein Peptide Lett. 1, 157-162]. In this study the alternative acetyl acceptor threonine and the alternative acyl donor, propionyl-CoA were used to further investigate the reaction mechanism of SAT from E. coli. Steady-state kinetic data and dead-end inhibition studies were again diagnostic of a random-order ternary complex reaction mechanism for alternative substrates. Since earlier kinetic studies with SAT from S. typhimurium suggested that cysteine competes with acetyl-CoA for binding, rather than serine with which it is isostructural, the specificity of the serine-binding pocket was assessed with three substrate mimics; beta-hydroxypropionic acid, glycine and ethanolamine. The data show that SAT interacts productively with the amino and hydroxymethyl moieties of serine, whereas the carboxyl group provides an essential contribution to binding strongly, supporting a view that cysteine will interact productively at the serine-binding site. Furthermore, since the hydroxymethyl contact region of the serine-binding site appears able to accommodate the methylene and acetyl moeties of threonine and O -acetyl-serine respectively, the site is unlikely to provide obligatory short-range contacts with the hydroxyl group of serine, a prerequisite for exclusion of cysteine. Such a proposal is supported by the results of micro-calorimetric studies which show that cysteine competes with serine for binding to SAT rather than with CoA. It follows that tight binding of cysteine at the serine-binding site near the catalytic centre may be the effector of a substantial reduction in the affinity of SAT for CoA, yielding the observed pattern of steady-state inhibition and the mechanism by which cysteine mediates effective end-product control of its synthesis.