The identification of a variant form of cystathionine beta-synthase in nematodes.

Characterization of the physical and catalytic properties of the enzyme responsible for nematode "activated L-serine sulfhydrase" activity (L-cysteine + R-SH-->cysteine thioether + H2S) has led to its identification as a novel, variant form (allelozyme) of cystathionine beta-synthase that is distinct from a mammalian-type synthase also present in nematodes. Additional work has demonstrated the ability of live Panagrellus redivivus to produce H2[35S] from exogenous L-[35S]cysteine and 2-mercaptoethanol, thus providing preliminary evidence for the in vivo operation of the activated L-serine sulfhydrase reaction in nematodes.     

Enzymatic hydrogen sulfide production in marine invertebrate tissues

At least some mammalian tissues produce H2S in vitro from L-cysteine at rates sufficient to have physiological effects. To determine whether tissues of macrofaunal invertebrates have the same capacity, we measured H2S production in tissue homogenates of the Manila clam Tapes philippinarum and the lugworm Arenicola marina. Tissue homogenates from both animals produced significant quantities of H2S gas upon addition of L-cysteine and the enzyme cofactor pyridoxal-5PRIME;-phosphate (10 mmol l(-1) and 2 mmol l(-1), respectively), while only tissues from T. philippinarum produced measurable H2S in the absence of added substrate or cofactor. In T. philippinarum tissues, H2S production was completely inhibited by the cystathionine beta-synthase (CBS) inhibitor aminooxyacetic acid (AOAA), suggesting that the majority of H2S production was via CBS pathways, while in A. marina body wall, AOAA inhibited only half of the total H2S production, indicating that the CBS pathway was not the only major source of H2S production. H2S production in tissues of T. philippinarum but not A. marina was doubled by the addition of a second thiol substrate (2.5 mmol l(-1) 2-mercaptoethanol), suggesting the presence of an 'activated serine sulfhydrase pathway', which had previously been demonstrated only in some microfauna.     

A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes

We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.     

Substrate inhibition of L-cysteine alpha,beta-elimination reaction catalyzed by L-cystathionine gamma-lyase of Saccharomyces cerevisiae

The alpha,beta-elimination of L-cysteine catalyzed by Saccharomyces cerevisiae L-cystathionine gamma-lyase (EC 4.4.1.1) was inhibited by the substrate. The absorption spectrum of the holoenzyme in the presence of L-cysteine showed that the substrate inhibition observed in this reaction was due mainly to removal of the cofactor.     

Biochemical characterisation of the enzyme responsible for "activated L-serine sulphydrase" activity in nematodes.

The biochemical properties of the enzyme responsible for nematode "activated L-serine sulphydrase" activity (L-cysteine + R-SH----cysteine thioether + H2S) have been investigated using primarily the gastro-intestinal nematodes Nippostrongylus brasiliensis and Haemonchus contortus. The activated L-serine sulphydrase enzyme was found to be cytosolic in origin and exhibited maximal activity at pH 9.0. Enzyme activity was widely distributed amongst the major tissues of adult female Ascaris suum but was particularly abundant in longitudinal muscle. The enzyme appeared to have a rigid specificity for L-cysteine as the primary thiol substrate, but was capable of utilising a number of sulphur amino acids (and derivatives) and nonphysiological thiols as second substrates. The best second thiol substrates were nonphysiological, hydroxyl-containing thiols that showed some structural similarity to the standard second substrate, 2-mercaptoethanol. Kinetic analyses revealed that the enzyme operates by a sequential catalytic mechanism, and the absolute Michaelis constants were: KL-cysteine = 0.21 +/- 0.02 mM and K2-mercaptoethanol = 5.58 +/- 0.59 mM. The enzyme was relatively insensitive to inhibition by a variety of substrate analogues and known inhibitors of pyridoxal 5'-phosphate dependent enzymes, whilst plant phenols caused significant levels of inhibition. The most potent inhibitors discovered were the anthelmintics bithionol, dichlorophene and hexachlorophene. Further characterisation revealed that hexachlorophene was a parabolic competitive inhibitor of the activated L-serine sulphydrase enzyme.     

Purification and Characterization of Cystathionine (gamma)-Lyase from Lactococcus lactis subsp. cremoris SK11: Possible Role in Flavor Compound Formation during Cheese Maturation

A cystathionine (gamma)-lyase (EC 4.4.1.1) ((gamma)-CTL) was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris SK11 by a procedure including anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The activity of SK11 (gamma)-CTL is pyridoxal-5(prm1)-phosphate dependent, and the enzyme catalyzes the (alpha),(gamma)-elimination reaction of L-cystathionine to produce L-cysteine, (alpha)-ketobutyrate, and ammonia. The native enzyme has a molecular mass of approximately 120 to 200 kDa and apparently consists of at least six identical subunits of 20 kDa. In this respect, the SK11 enzyme clearly differs from other bacterial cystathionine lyases, which are all tetrameric proteins with identical subunits of approximately 40 kDa. In addition, the specific activity of purified SK11 (gamma)-CTL toward L-cystathionine is relatively low compared with those reported for other bacterial cystathionine lyases. The SK11 enzyme shows a broad substrate specificity. In the case of L-methionine, the action of SK11 (gamma)-CTL results in the formation of methanethiol, a volatile sulfur compound known to be required in flavor development in cheddar cheese. The (alpha),(beta)-elimination reaction of L-cysteine is also efficiently catalyzed by the enzyme, resulting in the formation of hydrogen sulfide. Although the conditions are far from optimal, cystathionine (gamma)-lyase is still active under cheddar cheese-ripening conditions, namely, pH 5.0 to 5.4 and 5% (wt/vol) NaCl. The possible role of the enzyme in cheese flavor development is discussed.     

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.     

Enzymatic synthesis of L-cysteine

O-Acetylserine sulfhydrase in the form of a crude extract from Salmonella typhimurium LT2 was used for the production of L-cysteine from L-O-acetylserine and sodium hydrosulfide at pH 7.0 and 25 degrees C. The two substrates have quite different pH stability relationships. O-Acetylserine readily rearranges to N-acetylserine and the rate of this O --> N acyl transfer reaction increases at higher pH, temperature, and concentration of O-acetylserine. On the other hand, sodium hydrosulfide is more soluble at a higher pH. A stirred-tank bioreactor with a continuous substrate feed was employed to overcome this problem. The O-acetylserine feed was stored at its saturation level (2.05M) at pH 5.0, and the sodium hydrosulfide feed was dissolved at 2.05-2.3M without pH adjustment (pH >/= 11.5). Both substrates were simultaneously introduced into the bioreactor. The performance of the bioreactor was optimized by employing an automatic feedback control system to regulate the concentration of O-acetylserine in the bioreactor. This feedback control system was based on the fact that as the bioconversion proceeds, protons are produced along with cysteine. A pH controller thus detected the decrease in pH and activated the substrate pumps. After mixing in the bioreactor, these two substrate solutions behaved as a base due to the high alkalinity of sodium hydrosulfide. Thus, substrate infusion started when the pH was lower than the set point, i.e., the reaction pH, and stopped when the pH was raised higher than the set point. The amount of substrate introduced was determined by the alkalinity of the mixture of the two substrates, which in turn was controlled by the concentration of sodium hydrosulfide. After optimizing the sodium hydrosulfide concentration and the substrate feed rate, the bioconversion gave a productivity of 3.6 g L-cysteine/h/g dry cell weight S. typhimurium, an L-cysteine titer of 83 g/L and a molar yield based on O-acetylserine of 94%.     

Purification and properties of the membranal thiol oxidase from porcine kidney

A thiol oxidase was purified from porcine kidney cortex by chromatography of detergent-solubilized plasma membranes on cysteinylsuccinamidopropyl-glass beads, hydroxyapatite, and Sephacryl S-200. The oxidase was purified 2600-fold; 28% recovery of activity was obtained. With glutathione as substrate, the apparent Km was 0.73 mM and the V max was a 4.4 U/mg protein. The reaction catalyzed was 2 RSH + O2----RSSR + H2O2, and superoxide production was not detected during the reaction. Other low molecular weight thiols, including cysteine, dithiothreitol, N-acetylcysteine, and cysteamine, were substrates for the oxidase; 2-mercaptoethanol, reductively denatured ribonuclease A, and chymotrypsinogen A were not substrates. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band corresponding to 70 kDa; gel filtration on a Sephacryl column produced a single elution of activity with a protein corresponding to 120 kDa, indicating that the functional form is a dimer. On a high-pressure gel permeation column the protein eluted at 70 kDa under dilute conditions but at greater than 200 kDa at high concentrations, indicating that the protein also aggregates into larger multimers. Activity was inhibited by copper chelators, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), H2O2, and N-ethylmaleimide, suggesting the presence of copper and a sulfhydryl group at the active site. Following treatment with metal chelators, enzyme activity was reconstituted with CuSO4, but not with FeSO4. The purified enzyme contained 1 mol copper per subunit which was undetectable by electron paramagnetic resonance, suggesting that the copper is in a binuclear complex.     

Irreversible inhibition of jack bean urease by pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 25 degrees C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as L-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

酶法转化DL-ATC合成L-半胱氨酸的酶促反应条件研究

目的:考察酶源保存方式、酶促反应时间、底物pH值、底物浓度、酶浓度、金属离子等因素对酶活力的影响。方法:以假单胞菌(Pseudomonassp.)TS1138为供试菌株,采用酸式茚三酮法测定L-半胱氨酸含量,研究了酶法转化DL-ATC合成L-半胱氨酸的酶促反应条件。结果:TS1138菌株中L-半胱氨酸脱巯基酶具有较高的活性,而且Mg2 、Mn2 、Fe2 、Zn2 、Cu2 等5种金属离子对DL-ATC水解酶酶系有不同程度的抑制,其中Cu2 对该酶系的抑制作用很大。结论:确定了TS1138菌株酶法转化DL-ATC合成L-半胱氨酸的最适酶促反应条件,为酶促反应动力学的研究奠定了基础。     

Sodium nitroprusside potentiates hydrogen-sulfide-induced contractions in body wall muscle from a marine worm

Hydrogen sulfide (H2S) at concentrations of about 0.05 to 1 mmol.l(-1) appears to function as a gasotransmitter in vertebrates, analogous to nitric oxide (NO) and carbon monoxide, but the actions of H2S in invertebrate tissue have not been well studied. In this study, we investigated the role of H2S in modulating body wall muscle tone in the marine echiuran worm Urechis caupo (Echiuridae). We first determined that U. caupo body wall homogenates produce H2S upon addition of L-cysteine and pyridoxal-5'-phosphate (PLP), and that the rate is increased by addition of 2-mercaptoethanol, suggesting the presence of an activated L-serine sulfhydrase pathway. We then measured the contractile response of U. caupo body wall circular muscle strips to sodium hydrosulfide (NaHS)--which produces H2S in solution--and the NO donor sodium nitroprusside (SNP), both with and without subsequent application of acetylcholine (ACh). We found that NaHS alone stimulated contraction in muscle strips equivalent to about one-third the force of ACh alone, whereas SNP alone had no effect on muscle tone. However, simultaneous addition of NaHS with SNP elicited a much stronger contraction, reaching more than twice that of ACh alone, which could be increased further by subsequent application of ACh.     

Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus.

A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.     

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.     

Substrate specificity of cysteine lyase]

Substrate specificity is studied of cysteine lyase, a phosphopyridoxal-dependent enzyme belonging to the subgroup of beta-replacing lyases. This enzyme has a narrow specificity for the amino substrate; its only primary substrate is L-cysteine. Cysteine lyase has a broad specificity for the cosubstrate (replacing agent), catalysing the synthesis of L-cysteic acid from L-cysteine and sulfite ion or cystein thioesters (in the presence of some thiols). Enzyme is incapable to use alpha-phenyl- and alpha-methylcysteine as substrates. It is found that enzyme catalyses the exchange of alpha-H atoms of the aminoacid substrate cysteine with 3H2O. It does not catalyse alpha-hydrogenexchange in close structural analogues of substrate: L-alanine, D-serine, treonine, allo-threonine and 3-phosphoserine. L-Serine inhibited the synthesis of S-hydroxyethylcystein from cysteine and beta-mercaptoethanol (Ki of L-serine is 0,8-10(-2) M), participating at the first stage of reaction: the formation of a pyridoxylidenic derivative, which does not undergo the further alpha,beta-elimination of beta-replacement reactions.     

Studies on regulatory functions of malic enzymes. IV. Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli.

Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated. In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4). Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively. Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics. Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification. Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities. Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification. The Km values for the substrates and coenzymes were not significantly affected by NEM modification. Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents. Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol. Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed.     

Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation

Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.     

Anaerobic sulfatase-maturating enzymes, first dual substrate radical S-adenosylmethionine enzymes

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to C(alpha)-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S](2+,+) cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 '-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to C(alpha)-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

Alpha,beta-elimination reaction of O-acetylserine sulfhydrylase. Is the pyridine ring required?

O-Acetylserine sulfhydrylase (OASS) catalyzes the elimination of acetate from O-acetyl-L-serine (OAS) followed by addition of bisulfide to give L-cysteine. Site-directed mutagenesis has been used to replace the active site serine, S272, which forms a hydrogen bond to N1 of pyridoxal 5'-phosphate (PLP) with alanine and aspartate. Based on UV-visible spectral and steady-state kinetic studies, both mutant enzymes catalyze the elimination reaction with an efficiency equal to that of the wild-type enzyme. Data are consistent with an anti-E(2) reaction proposed for the elimination reaction.