The redox potentials and pH characteristics of d-amino-acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5±8.5 at 15 8C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two singleelectron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single-electron transfer potentials. The first electron potential is pH-independent, while the second electron transfer is pH-dependent exhibiting a < 260 mV/pH unit slope, consistent with a oneelectron/one-proton transfer. In the presence of the substrate analogue benzoate, the two-electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of E m vs. pH (230 mV/pH unit) corresponds to an overall two-electron/one-proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO±benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding.Keywords: flavoproteins; pH effect; redox properties; semiquinone stability.The enzyme d-amino-acid oxidase (EC 1.4.3.3; DAAO) catalyses the oxidation of the d-isomer of amino acids to a-imino acids and, after subsequent and spontaneous hydrolysis, to a-oxo acids and ammonia with a concomitant reduction of molecular oxygen to hydrogen peroxide. In the reductive half-reaction, the amino-acid substrate reduces the enzymebound FAD cofactor, producing reduced flavin and the imino acid. In the oxidative half-reaction, the reduced FAD±imino-acid complex reacts with molecular oxygen to form an oxidized FAD±imino-acid complex. The catalytic cycle is completed when the imino acid dissociates from the reoxidized enzyme and rapidly yields the corresponding a-oxo acid and ammonia.Three DAAOs have been obtained in a homogeneous form, from pig kidney, Rhodotorula gracilis, and Trigonopsis variabilis. In contrast with the well-known pig kidney DAAO (pkDAAO) [1], the two enzymes from yeast R. gracilis (RgDAAO) and T. variabilis (TvDAAO) are characterized by a much higher turnover number, good stability under a wide range of reaction conditions, and an active site that is sufficiently large to accommodate substrates even of considerable size [2,3]. Because of these properties, the yeast enzymes are widely used in biotechnological applications, e.g. for oxidation of cephalosporin C in the two-...
(1, 2). The precise mechanism of substrate dehydrogenation of this well studied enzyme (3) has not yet been solved, even if recently two groups have reported the crystal structure of the enzyme purified from pig kidney (pkDAAO) at a resolution of 2.6 and 3.0 Å, respectively (4, 5). Over the years, three main different mechanisms have been proposed for the reaction catalyzed by this flavoenzyme (see Mattevi et al. (6) for a recent review). (i) The hypothesis that the reductive half-reaction of DAAO involves the initial formation of a carbanion by abstraction of the ␣-H of the substrate as a proton comes from the elimination of halide from -chloro-D-alanine (7). (ii) The observation of a transfer of ␣-hydrogen of the substrate to the C(5) position of the enzyme reconstituted with 5-deaza-FAD provides evidence in favor of a direct hydride-transfer mechanism (8). (iii) Finally a concerted mechanism (consistent with the experimental evidence for a carbanion mechanism) in which ␣-H ϩ abstraction is coupled with the transfer of a hydride from the amino group of the substrate has been put forward (9).As a model for DAAO to have a better understanding of this crucial issue, we have used the enzyme from the red yeast Rhodotorula gracilis (RgDAAO). Actually, RgDAAO possesses peculiar properties, as the high catalytic efficiency and the tight binding with the coenzyme FAD, which distinguish it from the mammalian enzyme (10 -12). These properties are most probably related to its physiological role (yeast can metabolize D-amino acids and use them as the sole nitrogen and carbon source) and to an evolutionary drive. From comparison of the primary sequences of the known DAAOs, it is evident that only three residues, among those identified in or near the active site (13), are conserved (namely two tyrosines and one arginine). The presence of an arginine residue located at the active site and directly involved in DAAO catalysis was in fact previously proposed by various chemical modification studies (for a review, see Ref. 3 and references therein). The possibility that the arginine residue could be involved in substrate binding by electrostatic interaction with the substrate was inferred by Nishino et al. (14) from 2,3-butanedione modification of the mammalian enzyme, followed by reaction with dansylchloride. On the other hand, the reactivity with sulfite and the spectral properties of the native and cyclohexandione-modified pk-DAAO reconstituted with 8-mercapto-FAD suggested that the active site arginine could act as the positively charged group near the flavin N(1)-C(2)ϭO locus and responsible for stabilization of anionic flavin forms (15). This basic residue has been identified in the primary sequence of RgDAAO, by irreversible inhibition using phenylglyoxal (16), although the question regarding its role was not solved. More recently, the resolution of
The association between expression of the 67 kDa laminin receptor (67LR) and tumor aggressiveness has been convincingly demonstrated although the exact function of this molecule in the metastatic process has remained unclear. In this study, we tested whether the laminin-1, upon interaction with 67LR, promotes tumor cell aggressiveness; the investigation was based on: (i) the previous demonstration that soluble 67LR, as well as a 20-amino-acid peptide corresponding to the 67LR laminin binding site, changes the conformation of laminin upon interaction with this adhesion molecule and (ii) the known relevance of microenvironment remodeling by the tumor, leading to structural modification of extracellular matrix components in tumor progression. MDAMB231 breast carcinoma cells plated on peptide G-treated laminin-1 exhibited a polygonal array of actin filament bundles compared with cells seeded on native laminin-1 which presented the actin bundles organized as multiple cables parallel to margins. Furthermore, in cells seeded on peptide G-treated laminin-1, 67LR was distinct from the a6 integrin subunit in filopodia protrusions in addition to colocalizing with this integrin in focal adhesion plaques as it occurs when cells are plated on native laminin-1. In addition to differences in tumor cell adhesion and migration found in cells exposed to peptide G-treated vs native laminin-1, breast carcinoma cells seeded on modified laminin-1 showed a 6-fold increase in invasion capability compared with cells seeded on unmodified laminin-1. Alterations in actin organization as well as adhesion, migration and especially invasion observed in MDAMB231 cells in the presence of peptide G-treated laminin-1 were even found in MDAMB231 cells that, after selection for 67LR high expression, were seeded on native laminin-1. As the 67LR shedding is proportional to its expression level, these findings indicate a role for 67LR in changing laminin structure.Expression analysis of 97 genes encoding proteins that mediate cell matrix interactions, revealed significant differences between cells exposed to modified vs unmodified laminin-1 in 19 genes, 17 of which -including those encoding a3 integrin, extracellular matrix protein 1, proteolytic enzymes (such as MT1-MMP, stromelysin-3 and cathepsin L) and their inhibitors -were up-modulated in cells treated with modified laminin-1. Zymogram analysis clearly indicated a significant increase in the activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified laminin-1, without an increase in mRNA abundance as observed in microarray analysis. Invasiveness of tumor cells conditioned by modified laminin-1, evaluated as the capability to cross Matrigel basement, was significantly more inhibited by MMPinhibitor TIMP-2 than invasiveness induced by native laminin-1. Taken together, our findings indicate that the role of 67LR in tumor aggressiveness rests in its ability to modify laminin-1 thereby activating proteolytic enzymes that promote tumor cell invasion thr...
This poster provides an introduction to the use of infrared spcctmwpy in the investigation of enymaligand complexes. The enzyme a -C -. ontMrlingwitha substrate will form anacyl-bond to the ligand, wing a similar mechanism to some f3-htammcq which will show up in the infrared spectrum. The Sminc proteinases are good models for the d o n of the Pl actMl l l Ses, as they shere some of the same c d y t i c machinery and provide a relatively cheap and robust8ltmetive totheprohibitivdyacpensive B-lactamases diff;aarce !qwtmCopy hnnbeen used to w the acyl-bands
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