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Biomedical subjects

A Galdes

Publications and source records attributed to A Galdes.

34 records · Page 2Linked to original sources
PubMed1984

Cryospectrokinetic characterization of intermediates in biochemical reactions: carboxypeptidase A.

Cryospectrokinetic studies provide concurrent structural, kinetic, and chemical data on short-lived intermediates in the course of the interactions of enzymes with their substrates and of other, similar pairs of biomolecules. Subzero temperatures extend the lifetimes of these intermediates and, combined with rapid-mixing and rapid-scanning instrumentation, allow simultaneous measurement of both their physical-chemical and kinetic characteristics. For carboxypeptidase A, the spectra of a chromophoric, enzymatically functional cobalt atom at the active site signal the structure of the coordination complex during catalysis, while radiationless energy transfer between enzyme tryptophans and the fluorescent dansyl blocking group of rapidly hydrolyzed peptide and ester substrates provides the basis for measurement of the rates of formation and breakdown of intermediates. Subzero radiationless energy transfer kinetic studies of the zinc and cobalt enzymes disclose two intermediates in the hydrolysis of both peptides and esters and furnish all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2----E + P. The chemical and kinetic data indicate that neither of these is an acylenzyme intermediate. Both absorption and EPR spectra of the ES2 reaction intermediates consistently demonstrate the formation of transient metal complexes, differences between the effects induced by peptides and esters, and strong similarities between those induced by all peptides on the one hand and all esters on the other. The marked alterations of the cobalt spectra likely reflect the coordination of a substrate carboxyl and/or carbonyl group to the metal at a critical step in the course of catalysis. The cryospectrokinetic approach developed here in the mechanistic study of this metalloenzyme is applicable to the examination of transients of biochemical reactions in general. It will allow molecular characterization of previously elusive intermediates and greatly magnify the range of mechanistic questions that can be answered.

Animals↗
PubMed1980

The 1H nuclear-magnetic-resonance spectroscopy of cobalt(II)-beta-lactamase II.

The 1H n.m.r. spectra of beta-lactamase II in the presence of Co(II) were studied. Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II). The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II). The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E). However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences. A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues). This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme. The second site may be located on the surface of the molecule.

Apoenzymes↗
PubMed1980

Investigation of the structure of metallothioneins by proton nuclear magnetic resonance spectroscopy.

The proton nuclear magnetic resonance spectra of metallothioneins from horse, human, and sheep livers were investigated. The spectra of the metallothioneins from the three species are similar as are the two isoproteins from any one species. The spectra indicate that metallothioneins possess a well-defined tertiary structure. Zinc(II) and calcium(II) ions induce similar but not identical tertiary structures. Confirmatory evidence was obtained for the involvement of cysteine residues in metal binding, but no evidence was obtained for the involvement of any other amino acid residue in metal binding. The apoprotein thionein was found to exist essentially in a random-coil conformation with perhaps some residual segmental structure.

Animals↗
PubMed1980

A spectroscopic study of metal ion and ligand binding to beta-lactamase II.

beta-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted beta-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to beta-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4 degrees C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 microM (pH 6.0, 30 degrees C, 1 M NaCl) for the dissociation constant of Zn(II). The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.

Cephalosporinase↗
PubMed1978

Histidine residues of zinc ligands in beta-lactamase II.

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.

Apoenzymes↗
PubMed1978

The proton exchange of the pro-S hydrogen atom at C-1 in dihydroxyacetone phosphate and D-fructose 1,6-bisphosphate catalysed by class-I and class-II aldolases.

The efficacy of class-I and class-II aldolases in catalysing the C-1 proton exchange in fructose 1,6-bisphosphate and dihydroxyacetone phosphate was investigated. The rate of this reaction was at least two orders of magnitude slower in class-II than in the class-I aldolases. It is suggested that this difference reflects the formation of different intermediates in the reactions catalysed by the two classes of aldolase.

Dihydroxyacetone Phosphate↗
PubMedDate not supplied

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.

Alanine↗
PubMedDate not supplied

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.

Amino Acids↗