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

This work reports on the successful production of poly(ethylene furanoate) (PEF) thin films and a comparison of the enzymatic hydrolysis of PEF and poly(ethylene terephthalate) (PET) films with three different crystallinities (0, 10 and 20%).

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Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds

Canadian Journal of Biochemistry ◽

10.1139/o80-181 ◽

1980 ◽

Vol 58 (12) ◽

pp. 1335-1344 ◽

Cited By ~ 134

Author(s):

Nicholas E. Dixon ◽

Peter W. Riddles ◽

Carlo Gazzola ◽

Robert L. Blakeley ◽

Burt Zerner

Keyword(s):

Enzymatic Hydrolysis ◽

Mechanism Of Action ◽

Active Site ◽

Ph Dependence ◽

Jack Bean Urease ◽

Nickel Ion ◽

Jack Bean ◽

First Time ◽

Hydrolysis Of ◽

Hydrolysis Of Urea

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 °C, kcat has the values 5870, 85, 0.55, and 0.075 s−1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato–enzyme intermediate.

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

Biochemical Journal ◽

10.1042/bj2580765 ◽

1989 ◽

Vol 258 (3) ◽

pp. 765-768 ◽

Cited By ~ 7

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.

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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

The Journal of Organic Chemistry ◽

10.1021/jo00137a015 ◽

1982 ◽

Vol 47 (16) ◽

pp. 3105-3112 ◽

Cited By ~ 30

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

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Studies on N-acetylneuraminic acid aldolase

Biochemical Journal ◽

10.1042/bj1250275 ◽

1971 ◽

Vol 125 (1) ◽

pp. 275-284 ◽

Cited By ~ 20

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.

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

Biochemical Journal ◽

10.1042/bj3450315 ◽

2000 ◽

Vol 345 (2) ◽

pp. 315-319 ◽

Cited By ~ 4

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.

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Hydrolysis of Glycosyl Thioimidates by Glycoside Hydrolase Requires Remote Activation for Efficient Activity

Catalysts ◽

10.3390/catal9100826 ◽

2019 ◽

Vol 9 (10) ◽

pp. 826 ◽

Cited By ~ 3

Author(s):

Guillotin ◽

Assaf ◽

Pistorio ◽

Lafite ◽

Demchenko ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Active Site ◽

In Silico ◽

Glycoside Hydrolase ◽

Chemoenzymatic Synthesis ◽

Leaving Group ◽

Glycosyl Donor ◽

In Silico Modelling ◽

Leaving Group Ability ◽

Hydrolysis Of

Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or thioglycoligation. In both cases, the release of the thioimidoyl aglycone was remotely activated through a protonation driven by a carboxylic residue in the active site of the corresponding enzymes. A recombinant glucosidase (DtGly) from Dictyoglomus themophilum, previously used in biocatalysis, was also able to use such glycosyl thioimidates as substrates. Yet, enzymatic kinetic values analysis, coupled to mutagenesis and in silico modelling of DtGly/substrate complexes demonstrated that the release of the thioimidoyl moiety during catalysis is only driven by its leaving group ability, without the activation of a remote protonation. In the search of efficient glycosyl donors, glycosyl thioimidates are attractive and efficient. Their utility, however, is limited to enzymes able to promote leaving group release by remote activation.

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