scholarly journals Studies on N-acetylneuraminic acid aldolase

1971 ◽  
Vol 125 (1) ◽  
pp. 275-284 ◽  
Author(s):  
J. E. G. Barnett ◽  
D. L. Corina ◽  
G. Rasool
Keyword(s):  
Sodium Borohydride ◽  
Active Site ◽  
Half Life ◽  
Apparent Molecular Weight ◽  
Amino Acid Derivative ◽  
Enzyme Active Site ◽  
Acetylneuraminic Acid ◽  
Bivalent Metal Ions ◽  
Mercuric Ions ◽  
Hydrolysis Of

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.

scholarly journals A thiono-β-lactam substrate for the β-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate

1989 ◽  
Vol 258 (3) ◽  
pp. 765-768 ◽  
Author(s):  
B P Murphy ◽  
R F Pratt
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Direct Interaction ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Enzyme Active Site ◽  
Lactam Carbonyl ◽  
Beta Lactamase Ii ◽  
Hydrolysis Of

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.

Stereoselectivity in the epoxide hydrolase catalyzed hydrolysis of the stereoisomeric 3-tert-butyl-1,2-epoxycyclohexanes. Further evidence for the topology of the enzyme active site

1982 ◽  
Vol 47 (16) ◽  
pp. 3105-3112 ◽  
Author(s):  
Giuseppe Bellucci ◽  
Giancarlo Berti ◽  
Roberto Bianchini ◽  
Pasquale Cetera ◽  
Ettore Mastrorilli
Keyword(s):  
Active Site ◽  
Epoxide Hydrolase ◽  
Enzyme Active Site ◽  
Tert Butyl ◽  
Hydrolysis Of

scholarly journals Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

2000 ◽  
Vol 345 (2) ◽  
pp. 315-319 ◽  
Author(s):  
Dieter BECKER ◽  
Karin S. H. JOHNSON ◽  
Anu KOIVULA ◽  
Martin SCHÜLEIN ◽  
Michael L. SINNOTT
Keyword(s):  
Trichoderma Reesei ◽  
Active Site ◽  
Substrate Concentration ◽  
Initial Rate ◽  
Enzyme Active Site ◽  
Humicola Insolens ◽  
Cellobiohydrolase Ii ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Identical Crystal

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.

scholarly journals Neuraminidase inhibition by chemically sulphated glycopeptides

1979 ◽  
Vol 181 (2) ◽  
pp. 377-385 ◽  
Author(s):  
N Mian ◽  
C E Anderson ◽  
P W Kent
Keyword(s):  
Enzyme Inhibition ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Helix Pomatia ◽  
Submaxillary Gland ◽  
Competitive Inhibitor ◽  
Duodenal Mucosa ◽  
Dependent Manner ◽  
Enzyme Active Site ◽  
Acetylneuraminic Acid

Chemically sulphated glycopeptides (derived from pig duodenal mucosa) inhibited Clostridium perfringens neuraminidase (EC 3.2.1.18) activity in a pH-dependent manner. Analysis of inhibition kinetics data indicated that, although the enzyme inhibition could not be categorized into any of the classical types of inhibition, it could be interpreted as a function of the size and shape of the substrates used. The enzyme activity was inhibited by 86% and 40% when tested with bovine submaxillary-gland mucin (mol. wt. 4 x 10(5)-40 x 10(5) and N-acetylneuraminyl-lactose (mol. wt. 633) as substrates respectively. Presence of sulphated glycopeptide did not affect the binding of N-acetylneuraminic acid (mol. wt. 309), a competitive inhibitor of Vibrio cholerae neuraminidase, to the enzyme active site. The enzyme inhibition was thus considered to be due to steric hindrance as a consequence of the non-specific interactions between the enzyme molecule and polyanionic sulphated glycopeptide affecting the differential accessibility of the substrate molecules to the enzyme active site. The enzyme-inhibitor interaction could be suppressed by rapid and many-fold dilution of the reaction mixture, by concurrent addition of the inactive enzyme or by partial removal of the sulphate esters from the sulphated glycopeptide molecule by the action of Helix pomatia arylsulphatase (EC 3.1.6.1).

scholarly journals A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms

2020 ◽  
Vol 295 (52) ◽  
pp. 18379-18389
Author(s):  
James E. Stefano ◽  
Dana M. Lord ◽  
Yanfeng Zhou ◽  
Julie Jaworski ◽  
Joern Hopke ◽  
...  
Keyword(s):  
Immune Response ◽  
Monoclonal Antibodies ◽  
Tumor Microenvironment ◽  
Active Site ◽  
Effective Mechanism ◽  
Inactive Conformation ◽  
Enzyme Active Site ◽  
Multiple Partners ◽  
Hydrolysis Of ◽  
Tumor Eradication

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.

scholarly journals Enzymatic Hydrolysis of Polyester Thin Films at the Nanoscale: Effects of Polyester Structure and Enzyme Active-Site Accessibility

2017 ◽  
Vol 51 (13) ◽  
pp. 7476-7485 ◽  
Author(s):  
Michael Thomas Zumstein ◽  
Daniela Rechsteiner ◽  
Nicolas Roduner ◽  
Veronika Perz ◽  
Doris Ribitsch ◽  
...  
Keyword(s):  
Thin Films ◽  
Enzymatic Hydrolysis ◽  
Active Site ◽  
Enzyme Active Site ◽  
Hydrolysis Of

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
Keyword(s):  
Active Site ◽  
Reverse Reaction ◽  
Synthetic Route ◽  
Forward Reaction ◽  
Enzyme Active Site ◽  
Dead End ◽  
Effects Of Ph ◽  
Inhibition Studies ◽  
Hydrolysis Of ◽  
Substrate Concentrations

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.

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