scholarly journals A New Synthetic Route toN-Benzyl Carboxamides through the Reverse Reaction ofN-Substituted Formamide Deformylase

2013 ◽  
Vol 80 (1) ◽  
pp. 61-69
Author(s):  
Yoshiteru Hashimoto ◽  
Toshihide Sakashita ◽  
Hiroshi Fukatsu ◽  
Hiroyoshi Sato ◽  
Michihiko Kobayashi

ABSTRACTPreviously, we isolated a new enzyme,N-substituted formamide deformylase, that catalyzes the hydrolysis ofN-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A. 101:13726–13731, 2004,doi:10.1073/pnas.0405082101). Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizingN-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C1to C3), were active as acid substrates for the reverse reaction. Through this reaction,N-substituted carboxamides, such as NBFA,N-benzylacetamide, andN-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates.

1977 ◽  
Vol 167 (3) ◽  
pp. 831-833 ◽  
Author(s):  
G M Umezurike

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat in the absence or presence of added dioxan was found to be dependent on the ionization of two groups, which appeared to be a carboxyl group and an imidazole group. 2. Dioxan increased the Michaelis constant, Km, but decreased the maximum velocity, V.


1989 ◽  
Vol 258 (3) ◽  
pp. 765-768 ◽  
Author(s):  
B P Murphy ◽  
R F Pratt

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.


1982 ◽  
Vol 47 (16) ◽  
pp. 3105-3112 ◽  
Author(s):  
Giuseppe Bellucci ◽  
Giancarlo Berti ◽  
Roberto Bianchini ◽  
Pasquale Cetera ◽  
Ettore Mastrorilli

Author(s):  
Ying Fu ◽  
Jing-Yi Wang ◽  
Dong Zhang ◽  
Yu-Feng Chen ◽  
Shuang Gao ◽  
...  

A series of novel sulfonylurea benzothiazoline were designed by splicing active groups and bioisosterism. A solvent-free synthetic route was developed for the sulfonylurea benzothiazoline derivatives via the cyclization and carbamylation. All the compounds were characterized by IR, 1H-NMR, 13C-NMR, HRMS. The biological activity tests indicated the compounds could protect maize against the injury caused by chlorsulfuron to some extent. The molecular docking result showed that the new compound competed with chlorosulfuron to bind with the herbicide target enzyme active site to attain detoxification.


1971 ◽  
Vol 125 (1) ◽  
pp. 275-284 ◽  
Author(s):  
J. E. G. Barnett ◽  
D. L. Corina ◽  
G. Rasool

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37°C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent Ki for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive Ki 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the ∈-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.


2000 ◽  
Vol 345 (2) ◽  
pp. 315-319 ◽  
Author(s):  
Dieter BECKER ◽  
Karin S. H. JOHNSON ◽  
Anu KOIVULA ◽  
Martin SCHÜLEIN ◽  
Michael L. SINNOTT

We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.


2016 ◽  
Vol 14 (2) ◽  
pp. 361-367
Author(s):  
Le Thi Anh Tu ◽  
Le Ba Le

The bacterial enzyme, β-D-galactosidase, catalyzes the breakdown of the complex sugar lactose into its components - galactose and glucose-  simple sugars. The glycolysis of lactose by β-D-galactosidase is a point of attack for studies of the biochemical problem in disaccharide utilization and the genetic basis of enzyme constitution β-D galactosidase. The aim of the present study was to focus on optimization of a rapid enumeration method based on the enzymatic hydrolysis of 4-methylumbelliferyl-β-D-galactoside (MUGal) for toxicity test using Escherichia coli (E. coli) ATCC 8739 as a model. This rapid assay is based on the assumption that β-D-galactosidase is one marker for E. coli ATCC 8739. The enzymatic activity of E. coli ATCC 8739 was measured in a 25-minute assay. The effects of pH, temperature, nutrition, and substrate concentrations on enzyme activity were investigated. The enzyme β-D-galactosidase was shown to be induced with the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). The high level of production of β-D-galactosidase was found at bacteria incubated in tryptic soy broth without dextrose with IPTG. The optimum pH and temperature for β-D-galactosidase activity of E. coli ATCC 8739 was found to be 6.8 and 44.5 oC, respectively. The enzyme activity detected increased when raising the concentrations of the substrate (MUGal). The good linear correlation between logarithms of enzyme activities and CFU showed following supported the use of this method for toxicity experiments. Enzymatic methods and reference plate counts were significantly correlated. This method is sensitive, simple to perform and can be used as an alternative for traditional methods in detecting cell viability.


2020 ◽  
Vol 295 (52) ◽  
pp. 18379-18389
Author(s):  
James E. Stefano ◽  
Dana M. Lord ◽  
Yanfeng Zhou ◽  
Julie Jaworski ◽  
Joern Hopke ◽  
...  

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.


1987 ◽  
Vol 65 (6) ◽  
pp. 529-535 ◽  
Author(s):  
Gilles Cauet ◽  
Alain Friboulet ◽  
Daniel Thomas

The kinetics of the hydrolysis of butyrylthiocholine by horse serum butyrylcholinesterase (acylcholine acylhydrolase; BuChE; EC 3.1.1.8) exhibit an activation phenomenon at high substrate concentrations. At least two mechanistic models can account for the enzyme kinetics: one assumes the binding of an additional substrate molecule on the acyl–enzyme intermediate, and the other hypothesizes the existence of a peripheral regulatory site for the substrate. (1-Dimethylaminonaphthalene-5-sulfonamidoethyl)-trimethylammonium perchlorate, a potent reversible inhibitor, appears to affect BuChE activity by binding to a peripheral site. The inhibition is of the mixed type at low substrate concentrations and of the competitive type at high substrate concentrations. This is consistent with a peripheral site for the binding of the substrate responsible for the activation phenomenon.


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