Specificity of xanthine oxidase for nitrogen heteroaromatic cation substrates

1980 ◽  
Vol 58 (1) ◽  
pp. 49-57 ◽  
Author(s):  
John W. Bunting ◽  
Keith R. Laderoute ◽  
Donald J. Norris
Keyword(s):  
Xanthine Oxidase ◽  
Active Site ◽  
Hydrophobic Interactions ◽  
Substituent Effects ◽  
Steady State Kinetic ◽  
Bonding Interaction ◽  
Heteroaromatic Cation ◽  
Pyridinium Cations ◽  
Binding Orientations ◽  
State Kinetic

A variety of pyridinium, quinolinium, and benzoquinolinium cations have been investigated as potential substrates for milk xanthine oxidase at pH 9.9 and (or) pH 10.6. Steady-state kinetic parameters (kc, Km and (or) kc/Km) have been evaluated for all substrates which are enzymically oxidized. Simple N-alkyl pyridinium cations are neither substrates nor inhibitors, although N-aryl pyridinium cations are slowly oxidized to the 4-pyridinones. N-Methylpyridinium cations bearing 3-CONH2, 3-CONHCH3, 3-COCH3, 3-CO2− or 3-CN substituents are readily oxidized at C-6 and this suggests an important hydrogen-bonding interaction between an enzyme donor and the C-3 carbonyl substituent. A variety of N-methylquinolinium cations bearing C-6 substituents are enzymically oxidized at C-2. Analogous substituent effects on kc/Km for these 6-substituted 1-methylquinolinium cations and the corresponding 1-(substituted phenyl)-pyridinium cations is suggestive of the relative productive binding orientations of these two classes of substrate in the active site. N-Methyl benzoquinolinium and 1,10-phenanthrolinium cations are the best cationic substrates found to date, and suggest a relatively large active-site region for the reducing substrate, and important hydrophobic interactions between enzyme and substrate. The overall enzymic specificity observed for these cationic substrates allows a mapping of the general features of the reducing substrate binding site of this enzyme

scholarly journals Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its substrates

2003 ◽  
Vol 371 (2) ◽  
pp. 549-556 ◽  
Author(s):  
Maria Luisa DI PAOLO ◽  
Roberto STEVANATO ◽  
Alessandra CORAZZA ◽  
Fabio VIANELLO ◽  
Lorenzo LUNELLI ◽  
...  
Keyword(s):  
Active Site ◽  
Bovine Serum ◽  
Hydrophobic Interactions ◽  
Amine Oxidase ◽  
Amino Acid Residues ◽  
Deprotonated Form ◽  
Steady State Kinetic ◽  
Wide Range ◽  
Order Of Magnitude

A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4–10.2) and ionic strength (10–200mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region. Relatively small changes in kcat values (approx. one order of magnitude) accompanied by marked changes in Km and kcat/Km values (approx. six orders of magnitude) were observed. This behaviour was correlated with the presence of positively charged groups or apolar chains in the substrates. In particular, it was found that the docking of the physiological polyamines, i.e. spermidine and spermine, appears to be modulated by three amino acid residues of the active site, which we have named L-H+, G-H+ and IH+, characterized by pKa values of 6.2±0.2 [Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231–2239], 8.2±0.3 and 7.8±0.4 respectively. The electrostatic interaction between the protonated substrates and the enzyme containing the residues L-H+, G-H+ and IH+ in the deprotonated form, the on/off role of the IH+ residue and the role of hydrophobic interactions with substrates characterized by apolar chains are discussed.

Pre-Steady-State Kinetic Studies on the Glu171Gln Active Site Mutant of Adenosylcobalamin-Dependent Glutamate Mutase†

Biochemistry ◽  
2002 ◽  
Vol 41 (52) ◽  
pp. 15803-15809 ◽  
Author(s):  
Prashanti Madhavapeddi ◽  
David P. Ballou ◽  
E. Neil G. Marsh
Keyword(s):  
Steady State ◽  
Active Site ◽  
Kinetic Studies ◽  
Glutamate Mutase ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

Differences in the effects of pH on the hydrolytic and transgalactosylic reactions of β-galactosidase (Escherichia coli)

1983 ◽  
Vol 61 (4) ◽  
pp. 198-206 ◽  
Author(s):  
R. E. Huber ◽  
M. T. Gaunt ◽  
R. L. Sept ◽  
M. J. Babiak
Keyword(s):  
Rate Constant ◽  
Active Site ◽  
Kinetic Studies ◽  
Galactosidase Activity ◽  
Ph Optimum ◽  
Steady State Kinetic ◽  
Hydrolytic Reaction ◽  
Effects Of Ph ◽  
Rate Limiting ◽  
State Kinetic

Steady-state kinetic studies with β-galactosidase and various substrates were carried out to determine why the ratio of transgalactosylis to hydrolysis increased as a function of pH from 7.0 to 10.0. The rate constant [Formula: see text] for the formation of galactose (hydrolytic reaction) decreased whereas the rate constant (k4) for the transgalactosylic reaction (i.e., the formation of allolactose) remained constant. The equilibrium constant for acceptor dissociation from the galactosyl form of the enzyme was also unaffected by pH in the range studied; this was true whether the acceptor was glucose, sucrose, or glycerol. These results suggest that there is a group of high pKa at, or of influence at, the enzyme's active site which affects hydrolysis but not transgalactosylis. A further finding was that the rate constant for the breakage of the glycosidic bond decreased with pH in a manner different from the change observed for the hydrolytic rate constant (pKa 9.4 for glycosidic breakage as compared with 8.6 for hydrolysis). This could explain why the pH optimum for (β-galactosidase activity varies with substrate; different steps are rate limiting for different substrates.

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