Biomedical subjects

T N Wells

Publications and source records attributed to T N Wells.

133 records · Page 8Linked to original sources

PubMed1989

Protection of an unstable reaction intermediate examined with linear free energy relationships in tyrosyl-tRNA synthetase.

Linear free energy relationships (LFERs) are powerful tools in the search to understand the relationship between molecular structure and activity. They frequently link the changes in the rate constants for a reaction to changes in the equilibrium constant caused by alterations in structure. In physical-organic chemistry, these have been interpreted to give information on the structure of the transition state. Similar phenomena have been observed for reactions catalyzed by a series of engineered mutants of tyrosyl-tRNA synthetase from Bacillus stearothermophilus. LFERs are applied in this study to probe how the enzyme minimizes its side reactions. A linear free energy relationship is shown between the binding of the unstable enzyme-tyrosyl adenylate complex and its rate constant of hydrolysis. However, mutations of a key residue, His48, show significant deviation from the relationship, implying a role for the side chain in protection of the complex from hydroxide attack. A second linear free energy relationship is shown linking the rate and equilibrium constants for tyrosyl adenylate binding to the enzyme. Four distinct classes of mutation are discussed in the context of this relationship. The data from all but one of these groups of mutations conform well to a linear free energy relationship between the dissociation rate and dissociation equilibrium constants for the enzyme-tyrosyl adenylate complex with slope beta = 1.01 +/- 0.08. The specificity of binding of tyrosyl adenylate is determined solely by its dissociation rate constant of the intermediate, and the mutations have relatively little effect on the enzyme-tyrosyl adenylate association rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1987

Structure-activity relationships in engineered proteins: analysis of use of binding energy by linear free energy relationships.

The activity of mutant enzymes can be analyzed quantitatively by structure-activity relationships in a manner analogous to Brønsted or Hammett plots for simple organic reactions. The slopes of such plots, the beta values, indicate for the enzymatic reactions the fraction of the overall binding energy used in stabilizing particular complexes. In particular, information can be derived about the interactions between the enzyme and the transition state. The activities of many mutant tyrosyl-tRNA synthetases fit well simple linear free energy relationships. The formation of enzyme-bound tyrosyl adenylate (E.Tyr-AMP) from enzyme-bound tyrosine and ATP (E.Tyr-ATP) results in an increase in binding energy between the enzyme and the side chain of tyrosine and the ribose ring of ATP. Linear free energy plots of enzymes mutated in these positions give the fraction of the binding energy change that occurs on formation of the transition state for the chemical reaction and the various complexes. It is shown that groups that specifically stabilize the transition state of the reaction are characterized by beta values much greater than 1. This is found for residues that bind the gamma-phosphate of ATP (Thr-40 and His-45) and have previously been postulated to be involved in transition-state stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗

PubMed1986

Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases.

The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus. The formation constant (=[Tyr-AMP] [PPi]/[ATP] [Tyr]) at pH 7.78, 25 degrees C, and 10 mM MgCl2 is (3.5 +/- 0.5) X 10(-7). This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25 degrees C of -16.7 kcal mol-1. All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex. This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution. These are technically difficult measurements because the dissociation constants are so low and the complexes unstable. The task was simplified by using mutants prepared by site-directed mutagenesis. These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes. The dissociation constants were in the range (3.5-38) X 10(-12) M, with that for wild type at 13 X 10(-12) M. The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution. This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes.

Adenosine Monophosphate↗

PubMed1986

Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase.

The utilization of enzyme-substrate binding energy in catalysis has been investigated by experiments on mutant tyrosyl-tRNA synthetases that have been generated by site-directed mutagenesis. The mutants are poorer enzymes because they lack side chains that form hydrogen bonds with ATP and tyrosine during stages of the reaction. The hydrogen bonds are not directly involved in the chemical processes but are at some distance from the seat of reaction. The free energy profiles for the formation of enzyme-bound tyrosyl adenylate and the equilibria between the substrates and products were determined from a combination of pre-steady-state kinetics and equilibrium binding methods. By comparison of the profile of each mutant with wild-type enzyme, a picture is built up of how the course of reaction is affected by the influence of each side chain on the energies of the complexes of the enzyme with substrates, transition states, and intermediates (tyrosyl adenylate). As the activation reaction proceeds, the apparent binding energies of certain side chains with the tyrosine and nucleotide moieties increase, being weakest in the enzyme-substrate complex, stronger in the transition state, and strongest in the enzyme-intermediate complex. Most marked is the interaction of Cys-35 with the 3'-hydroxyl of the ribose. Removal of the side chain of Cys-35 leads to no change in the dissociation constant of ATP but causes a 10-fold lowering of the catalytic rate constant. It contributes no net apparent binding energy in the E X Tyr X ATP complex and stabilizes the transition state by 1.2 kcal/mol and the E X Tyr-AMP complex by 1.6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗

PubMed1983

Bromocresol purple and the measurement of albumin. Falsely high plasma albumin concentrations eliminated by increased reagent ionic strength.

This study was undertaken to investigate the extent of the reported discrepancy between serum and plasma albumin concentrations measured by the bromocresol purple (BCP) method. We have shown that plasma albumin concentrations are falsely increased by turbidity due to the precipitation of fibrinogen when plasma is diluted into the BCP reagent. Modification of the BCP buffer (to 0.1M acetate containing 0.15M NaCl, pH 5.5) eliminates the turbidity and thus permits the measurement of albumin in either serum or plasma. Plasma albumin concentrations measured with the modified BCP reagent show excellent correlation with results by an immunoturbidimetric method.

Bromcresol Purple↗

PubMedDate not supplied

The CC chemokine receptor antagonist met-RANTES inhibits eosinophil effector functions.

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine-induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met-RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP-3 and eotaxin was investigated. Met-RANTES had no intrinsic activity on [Ca2+]i transients in eosinophils and was able to dose-dependently inhibit [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Besides its effect on [Ca2+]i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP-3 and eotaxin. The results of this study lead to the conclusion that Met-RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.

Calcium↗

PubMedDate not supplied

Aminooxypentane-RANTES induces CCR3 activation and internalization of CCR3 from the surface of human eosinophils.

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca(2+)](i)) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37 degrees C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.

Cells, Cultured↗