Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Stereospecificity of plant microsomal cinnamic acid 4-hydroxylase

Cinnamic acid 4-hydroxylase from parsley cell suspension cultures is specific for trans-cinnamic acid as substrate. cis-Cinnamic acid is a competitive inhibitor of the enzyme with a K_i of approximately 0.34 mm. Hydrocinnamic acid is neither a substrate nor an inhibitor.     

Diffusional and electrostatic effects on apparent kinetic parameters of reactions catalyzed by enzyme immobilized on the external surface of a support

Diffusional and electrostatic effects on the apparent maximum reaction rate V_m^app and the apparent Michaelis constant K_m^app were investigated theoretically for a system in which an enzyme immobilized on the external surface of a solid support catalyzes a reaction according to Michaelis-Menten kinetics. In such a system, the dependence of V_m^app and K_m^app on the substrate concentration can be expressed analytically. When the support and substrate carry charges of the same sign, resulting in a repulsive force between them, both V_m^app and K_m^app decrease with increasing substrate concentration, but they never decrease below the respective intrinsic values. On the other hand, when the support and substrate carry charges of opposite sign and therefore an attractive force occurs, V_m^app decreases towards its intrinsic value, while K_m^app decreases to values below its intrinsic value in the region of high substrate concentration.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

A convenient analysis of Michaelis enzyme kinetic progress curves based on second derivatives

A simple method is described for the estimation of the Michaelis parameters, K_m and V_m, from a single progress curve at a single substrate concentration without the need to follow the reaction to completion. By measuring the substrate concentration and the time when the second derivative is at a minimum, K_m and V_m can be easily obtained.     

Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules

Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent K_m (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10⁻⁵ M, which is compared to 3 × 10⁻³ M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent K_m estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction.     

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis,cis-muconic acid production

This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K _m and V _max was 12.8 μM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 μM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.     

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula: ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE,LAVANDULYL DIPHOSPHATE SYNTHASE*

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K_m and k_cat values of 208 ± 12 μm and 0.1 s⁻¹, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.     

Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Adducts formed between deoxyguanosine and cis-Dichlorodiammineplatinum(II): Separation by means of cation-exchange chromatography

The interaction between deoxyguanosine (dG) and cis-dichlorodiammineplatinum(II) (cis-Pt) leads to the 2:1 and the 1:1 dG-Pt adducts. These adducts were separated on an Aminex A6 cationexchange column by use ot 0.01 M K₂CO₃ (pH 11) as an eluent. The stoichiometry of the adducts was determined from the ^195mPt radioactivity and from the absorbance of the guanine chromophore at 280 nm. Time-course studies show that dG reacts initially with cis-Pt to form the 1:1 adduct, which then interacts with a second molecule of dG to form the 2:1 adduct. Acid hydrolysis (100°C in 88% formic acid for 5–15 min) of the 1:1 and 2:1 adducts results in their conversion to two new products, which elute differently from the column but which still contain Pt bound in the same stoichiometric ratio to dG as in the nonhydrolyzed adducts. The hydrolyzed adducts show a negative diphenylamine reaction indicative ot cleavage of the glycosidic bond. It is concluded that mild acid hydrolysis converts the 1:1 and 2:1 dG-Pt adducts into the corresponding guanine-Pt adducts, which are chromatographically distinguishable. This acid hydrolysis-high pressure liquid chromatography (HPLC) procedure has application to the identification of the Pt adducts formed in DNA.     

The specific,submicromolar-Km ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein,including a selectivity for Mn as activating cation and increase in Km for ADP-ribose,both elicited by H2O2

Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low-K_m ADP-ribose pyrophosphatase. In humans, a submicromolar-K_m ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 10²–10³ higher K_m. Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar K_m for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg²⁺ or Mn²⁺ as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H₂O₂ alters the Mg²⁺/Mn²⁺ responses and increases the K_m values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose.     

Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD⁺, is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine’s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V _max , K _cat , and K _m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.     

Mono- and diphenolase activity from fruit of Malus pumila

Apple fruit used for beverage production has a polyphenol oxidase which does not hydroxylate tyrosine under any conditions but it hydroxylates p-coumaric acid in the presence of NADH, and phloridzin in the absence of cofactors. The apparent K_ms for hydroxylation of phloridzin and p-coumaric acid are 1.5 and 4 mM, respectively. However, subsequent oxidation of 3-hydroxyphloridzin or caffeic acid has an apparent K_m of 200 nM. The oxidation products of 3-hydroxyphloridzin are complex and a stable dimeric quinone is finally formed. The apparent K_ms for oxidation of catechin, epicatechin, chlorogenic acid, l-Dopa and 4-methyl catechol are 4.7, 5.7, 6.0, 2.7 and 3.2 mM, respectively. The K_m for oxygen was 4.3 % although there was marked substrate inhibition by oxygen above 30 %. Polyphenol oxidase was stable at pH 3.5–4.5 with an optimum of 4.5.     

Metabolism of sphingadiene and characterization of the sphingadiene-producing enzyme FADS3

Of the long-chain bases (LCBs) that comprise the ceramides (CERs) present in mammals, only 4,14-sphingadiene (sphingadiene; SPD) has a cis double bond (at C14). Because of this unique structure, the metabolism of SPD may differ from that of other LCBs, but whether this is the case remains unclear. FADS3 is responsible for introducing the cis double bond in SPD. However, the substrate specificity of FADS3 and cofactors involved in the FADS3-catalyzed reaction are also unknown. In the present study, a cell-based assay using a ceramide synthase inhibitor and an in vitro experiment showed that FADS3 is active toward sphingosine (SPH)-containing CERs (SPH-CERs) but not toward free SPH. FADS3 exhibits specificity with respect to the chain length of the SPH moiety of SPH-CERs (active toward C16–20), but not that of the fatty acid moiety. Furthermore, FADS3 is active toward straight-chain and iso‐branched-chain SPH-containing CERs but not toward anteiso-branched forms. In addition to SPH-CERs, FADS3 also shows activity toward dihydrosphingosine-containing CERs, but this activity is approximately half of that toward SPH-CERs. It uses either NADH or NADPH as an electron donor, and the electron transfer is facilitated by cytochrome b₅. The metabolic flow of SPD to sphingomyelin is predominant over that to glycosphingolipids. In the metabolic pathway from SPD to fatty acids, the chain length of the SPD is reduced by two carbons and the trans double bond at C4 is saturated. This study thus elucidates the enzymatic properties of FADS3 and the metabolism of SPD.     

Localization of ATPase protein in sarcoplasmic reticulum membrane.

The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p-Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4′-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys.169, 84–90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p-hydroxybenzalacetone. The apparent K_m values for malonyl-CoA and p-coumaroyl-CoA in the reaction leading to naringenin, and for p-coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 μm, respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p-methoxycinnamoyl-CoA, isoferuloyl-CoA, or p-hydroxybenzoyl-CoA.None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo. The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p-coumaroyl-CoA.     

The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide by thermolysin

The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the K_m for the enzyme substrate interaction is 2.5 × 10^?3m at pH 7.2. This K_m is an order of magnitude less than what has been previously assumed to be the K_m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10^?3m substrate and is based on the assumption that the substrate concentration is much less than the K_m. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10^?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K_m.