scholarly journals Interactions between inhibitors of dihydrofolate reductase

1989 ◽  
Vol 258 (2) ◽  
pp. 335-342 ◽  
Author(s):  
K Bowden ◽  
A D Hall ◽  
B Birdsall ◽  
J Feeney ◽  
G C K Roberts
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Dihydrofolate Reductase ◽  
Binding Sites ◽  
High Field ◽  
Ph Dependence ◽  
Histidine Residue ◽  
Adenine Ring ◽  
Steady State Kinetics ◽  
Pka Value

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be ‘tightly binding’ inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a ‘sulphonamide-binding site’ on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

scholarly journals The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

1993 ◽  
Vol 290 (1) ◽  
pp. 15-19 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Chemical Modification ◽  
Ph Dependence ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Histidine Residue ◽  
Pka Values ◽  
Primary Hydroxy Group ◽  
Pka Value ◽  
Catalytic Role ◽  
Kinetics Of

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Catalysis of potato epoxide hydrolase, StEH1

2005 ◽  
Vol 390 (2) ◽  
pp. 633-640 ◽  
Author(s):  
Lisa T. Elfström ◽  
Mikael Widersten
Keyword(s):  
Steady State ◽  
Epoxide Hydrolase ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Kinetic Mechanism ◽  
Kinetic Measurements ◽  
Steady State Kinetics ◽  
Substrate Conversion ◽  
Km Value ◽  
The Individual

The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 μmol·min−1·mg−1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300→Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

scholarly journals pH-dependence of warfarin binding to α1-acid glycoprotein (orosomucoid)

1993 ◽  
Vol 289 (3) ◽  
pp. 767-770 ◽  
Author(s):  
S Urien ◽  
F Brée ◽  
B Testa ◽  
J P Tillement
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Ph Dependence ◽  
Protonated Form ◽  
Association Constants ◽  
Histidine Residue ◽  
Difference Spectra ◽  
Acid Glycoprotein ◽  
Binding Data ◽  
Decreasing Ph

The binding of warfarin to alpha 1-acid glycoprotein (AAG) was found to increase with decreasing pH. The u.v.-visible difference spectra generated upon binding to AAG at pH 5.0 or 7.4 showed warfarin to bind as the anion. Warfarin-binding data were satisfactorily fitted to a model that incorporates the effect of pH and discriminates the association constants of the non-protonated and protonated binding site of the protein. It was shown that AAG-binding site in the protonated form had a markedly higher affinity for warfarin than the non-protonated form, with a pK value of 7.7 +/- 0.1, which is likely to be a histidine residue. Among other possible interactions, it is suggested that ligand binding to AAG involves a reinforced hydrogen bond.

scholarly journals Histidine residues of zinc ligands in β-lactamase II

1978 ◽  
Vol 175 (2) ◽  
pp. 441-447 ◽  
Author(s):  
G S Baldwin ◽  
A Galdes ◽  
H A O Hill ◽  
B E Smith ◽  
S G Waley ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Binding Site ◽  
Binding Sites ◽  
Zinc Binding ◽  
Histidine Residue ◽  
Beta Lactamase ◽  
Histidine Residues ◽  
Zinc Enzyme ◽  
Zinc Binding Site ◽  
Ph Range

1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0–9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

scholarly journals A novel 1,4-dihydropyridine-binding site on mitochondrial membranes from guinea-pig heart, liver and kidney

1988 ◽  
Vol 253 (1) ◽  
pp. 49-58 ◽  
Author(s):  
G Zernig ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Binding Site ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Capacity ◽  
Mitochondrial Membranes ◽  
Guinea Pig Heart ◽  
Heat Stable ◽  
Liver And Kidney ◽  
Dihydropyridine Binding Site

The 1,4-dihydropyridine (+/-)-[3H]nitrendipine reversibly binds to mitochondrial preparations from guinea-pig heart with a dissociation constant (Kd) of 593 +/- 77 nM and a maximum density of binding sites (Bmax.) of 1.75 +/- 0.27 nmol/mg of protein. This low-affinity high-capacity 1,4-dihydropyridine-binding site does not discriminate between the enantiomers of nitrendipine and is also found in mitochondrial membranes from guinea-pig liver (Kd 586 +/- 91 nM; Bmax. 0.36 +/- 0.04 nmol/mg of protein) and kidney (Kd 657 +/- 149 nM; Bmax. 0.56 +/- 0.12 nmol/mg of protein). Phenylalkylamines (e.g. verapamil) inhibit (+/-)-[3H]nitrendipine binding with micromolar inhibition constants, but the benzothiazepine D-cis-diltiazem, a potent Ca2+-channel blocker, is without effect. The binding is heat-stable, shows a V-shaped pH-dependence with a minimum around pH 7.0, and is strongly dependent on ionic strength in the incubation medium. The cations La3+ greater than Cd2+ much greater than Co2+ greater than Ca2+ much greater than Ba2+ greater than Mg2+ greater than Li+ greater than Na+ and the anions NO3- greater than C1- greater than or equal to F- stimulate the binding, whereas PO4(3-) greater than SO4(2-) slightly inhibit it. The low-affinity (+/-)-[3H]nitrendipine-binding site located on the mitochondrial inner membrane is biochemically and pharmacologically different from the 1,4-dihydropyridine-receptor domain of the L-type Ca2+ channel. Furthermore, it is not identical with any of the low-affinity 1,4-dihydropyridine-binding sites described so far.

scholarly journals Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion

2003 ◽  
Vol 372 (3) ◽  
pp. 735-746 ◽  
Author(s):  
Syeed HUSSAIN ◽  
Surapong PINITGLANG ◽  
Tamara S. F. BAILEY ◽  
James D. REID ◽  
Michael A. NOBLE ◽  
...  
Keyword(s):  
Steady State ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Catalytic Site ◽  
Rate Determining Step ◽  
Steady State Kinetic ◽  
Pka Value ◽  
Site Function ◽  
Hydrolysis Of ◽  
State Kinetic

The acylation and deacylation stages of the hydrolysis of N-acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential pKa values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp161 is the source of a key electrostatic modulator (pKa 5.0±0.1) in actinidin, analogous to Asp158 in papain, whose influence is not detected kinetically; it is always in the ‘on’ state because of its low pKa value (2.8±0.06).

Automated site-directed drug design : the formation of molecular templates in primary structure generation

1989 ◽  
Vol 236 (1283) ◽  
pp. 141-162 ◽  
Keyword(s):  
Binding Site ◽  
Dihydrofolate Reductase ◽  
Binding Sites ◽  
Distance Matrix ◽  
Ligand Binding Site ◽  
Structure Generation ◽  
Molecular Graphs ◽  
Natural Ligands ◽  
Optimization Routine ◽  
Accessible Surface

In this paper the spacer skeleton concept is used to produce molecular graphs of putative ligands for binding sites. The skeletons are transformed into molecular templates within the constraints of the accessible surface of the ligand-binding site. A distance-matrix method is used to compare ligand points with vertices of the spacer skeleton through a permutation of all possible correspondences. A tolerance parameter is used to screen for poor matches. As a result, a small number of matched vertices and ligand points are produced. These are fitted into the site by a constrained optimization routine using an analytical function. Ligand points fall within the site and are optimally positioned adjacent to the corresponding site points; other vertices of the spacer skeleton lying beneath the accessible surface of the site are clipped off. A molecular template is thereby formed with its vertices linked to the ligand points. The final step is to verify that the bonding integrity of the skeleton remains. The computational methods outlined in this paper have been tested at two binding sites: the pteridine binding site in dihydrofolate reductase and the amidinophenylpyruvate site of trypsin. Molecular graphs for both sites were generated automatically; they showed strong similarity to those of the natural ligands.

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