scholarly journals Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase

1981 ◽  
Vol 199 (3) ◽  
pp. 599-602 ◽  
Author(s):  
B Franzen ◽  
C Carrubba ◽  
D S Feingold ◽  
J Ashcom ◽  
J S Franzen
Keyword(s):  
Glucose Dehydrogenase ◽  
Thick Layer ◽  
Bovine Liver ◽  
Thiol Group ◽  
Paper Electrophoresis ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Diphosphate Glucose ◽  
Adsorption Desorption

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.

scholarly journals Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 701-704 ◽  
Author(s):  
J S Franzen ◽  
P Marchetti ◽  
R Ishman ◽  
J Ashcom
Keyword(s):  
Catalytic Activity ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Thiol Group ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Irreversible Denaturation ◽  
Diphosphate Glucose ◽  
Uridine Diphosphate Glucose Dehydrogenase

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.

scholarly journals The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

1974 ◽  
Vol 141 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Paul A. Gainey ◽  
Charles F. Phelps
Keyword(s):  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Uridine Diphosphate ◽  
Protein Fluorescence ◽  
Diphosphate Glucose ◽  
Ph Dependent ◽  
Uridine Diphosphate Glucose Dehydrogenase ◽  
Biphasic Profile

The binding of NAD+and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD+and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3μm. The binding of NAD+to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60–70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.

A DFT study on the catalytic mechanism of UDP-glucose dehydrogenase

2010 ◽  
Vol 88 (8) ◽  
pp. 804-814 ◽  
Author(s):  
WenJuan Huang ◽  
Jorge Llano ◽  
James W. Gauld
Keyword(s):  
Pathogenic Bacteria ◽  
Catalytic Mechanism ◽  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Polarizable Continuum Model ◽  
Uridine Diphosphate ◽  
Reaction Field ◽  
Enzyme Active Site ◽  
Site Model ◽  
Diphosphate Glucose

Uridine 5′-diphosphate glucuronic acid (UDPGlcUA) is a key intermediary metabolite in many species, including pathogenic bacteria and humans. It is biosynthesized from UDP-glucose (UDPGlc) by uridine diphosphate glucose dehydrogenase (UDPGlcDH) via a twofold two-electron–one-proton oxidation that successively transforms the 6-hydroxymethyl of glucopyranose into a formyl, and the latter into the final carboxylic function. The catalytic mechanism of UDPGlcDH was investigated using a large enzyme active-site model in combination with the B3LYP method and the polarizable continuum model (IEF-PCM) self-consistent reaction field. The latter was used to correct for the long-range electrostatic effect of the protein environment. The overall mechanism consists of four catalytic steps: (i) NAD+-dependent oxidation of glucose to glucuronaldehyde, (ii) nucleophilic addition of Cys260–SH to glucuronaldehyde to form a 6-thiohemiacetal intermediate, (iii) NAD+-dependent oxidation of the 6-thiohemiacetal to form a 6-thioester intermediate, and finally, (iv) hydrolysis of the 6-thioester to give glucuronic acid. In addition, this study also provides insight into the debated roles of Lys204 and Asp264, and the most likely protonation state of a reactive Michaelis complex of UDPGlcDH.

scholarly journals The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane

1988 ◽  
Vol 249 (1) ◽  
pp. 239-245 ◽  
Author(s):  
R R Ramsay
Keyword(s):  
Mouse Liver ◽  
Bovine Liver ◽  
Thiol Group ◽  
Carnitine Palmitoyltransferase ◽  
Native Enzyme ◽  
Thiol Groups ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Chain Acyl ◽  
Carnitine Acyltransferases

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5′-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5′-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.

scholarly journals A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1972 ◽  
Vol 129 (4) ◽  
pp. 821-830 ◽  
Author(s):  
P. A. Gainey ◽  
T. C. Pestell ◽  
C. F. Phelps
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Peptide Mapping ◽  
Ph Dependence ◽  
Glucose Dehydrogenase ◽  
Group Analysis ◽  
Subunit Structure ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Thiol Reactivity

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ‘mapping’ in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). 5. NAD+, UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5′-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4–8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.

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