PubMed Health⌕ Search

Biomedical subjects

A J Wilkinson

Publications and source records attributed to A J Wilkinson.

88 records · Page 5Linked to original sources
PubMed1985

Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase.

Residue Thr-51 at the active site of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) has been replaced with all the smaller amino acids by protein engineering to investigate direct and indirect effects of mutation on substrate binding and catalysis. The gamma-hydroxyl group of Thr-51 was thought to be 0.5 A too far from the ribose ring oxygen of ATP to form a hydrogen bond. Consistent with this, it is found that mutation of Thr-51----Cys-51, which should place the gamma-thiol group within its correct distance for hydrogen bonding, increases the affinity of the enzyme for ATP. Other mutations (Ser-51, Ala-51, and Gly-51) show the contributions to binding of the other atoms in the side chain of Thr-51. A family of enzymes has been produced, TyrTS(Thr-51) (wild type), TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51), in which the value of kcat/KM for ATP in aminoacylation increases along the series. This is achieved by the value of KM decreasing significantly (2.5, 1.25, 0.29, and 0.019 mM, respectively) while there are smaller decreases in kcat (4.7, 4.0, 2.9, and 1.8 s-1, respectively). These variations cause each one of the enzymes to be more active than the others at particular concentrations of ATP. For example, at concentrations of ATP greater than 5.9 mM, TyrTS(Thr-51) is the most active, while TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51) are the most active at 5.9-2.2, 2.2-0.42, and less than 0.42 mM ATP, respectively. Interestingly, position 51 shows variation in tyrosyl-tRNA synthetases isolated from different organisms.

Adenosine Triphosphate↗
PubMed1985

Probing histidine-substrate interactions in tyrosyl-tRNA synthetase using asparagine and glutamine replacements.

We have analyzed the interactions of a histidine residue with a substrate using site-directed mutagenesis. Previous studies on tyrosyl-tRNA synthetase from Bacillus stearothermophilus have shown that a histidine residue (His-48) makes an interaction with ATP, which is improved on mutating Thr-51----Pro-51. We find on replacing His-48 in wild-type enzyme with either asparagine or glutamine that Asn-48 is equally as good as His-48 but His-48----Gln-48 leads to a far lower activity. The side chain of an asparagine residue may be superimposed on that of a histidine so that the amide-NH2 group of asparagine occupies the same position as the pi-N of histidine, whereas the equivalent -NH2 group of glutamine may be superimposed upon the tau-N. This suggests that it is the pi-N of histidine that hydrogen bonds with ATP and that there is no significant electrostatic interaction between the histidine and ATP. Incorporating the Pro-51 mutation into each of the Asn-48 and Gln-48 mutants gives an improvement in the affinity of the enzyme for ATP, but this improvement is less than that seen with the wild-type enzyme.

Amino Acid Sequence↗
PubMed1984

The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus).

In a previous study, a mutant of tyrosyl-tRNA synthetase in which a threonine residue (Thr51) was converted to proline dramatically improved the affinity of the enzyme for its ATP substrate. How does Pro51 improve the enzyme's affinity for ATP? A priori, Pro51 might interact directly with the ATP, or it might distort the polypeptide backbone and thereby force new or improved contacts elsewhere from the enzyme to ATP. By making mutants of the Pro51 enzyme at two residues that make hydrogen bonds to the ATP substrate, we show that Pro51 greatly improves the strength of one of these contacts. Thus the propagation of a structural change in an enzyme induced by mutation may be detected by the introduction of further mutations.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation.

Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.

Amino Acid Sequence↗
PubMed1983

Deletion mutagenesis using an 'M13 splint': the N-terminal structural domain of tyrosyl-tRNA synthetase (B. stearothermophilus) catalyses the formation of tyrosyl adenylate.

The X-ray crystallographic structure of tyrosyl-tRNA synthetase (TyrTS) comprises only the N-terminal 320 amino acids of the molecule as the C-terminal 99 amino acids are poorly ordered in the crystal. A new technique, employing a single-stranded M13 splint, has been used to direct a deletion in the cloned gene of TyrTS so as to remove the disordered C-terminal region. We find that the truncated enzyme catalyses the formation of tyrosyl adenylate with unchanged Kcat and Km values and the crystallographic model must therefore include all the binding and catalytic residues involved in tyrosine activation. However, the truncated enzyme no longer binds tRNATyr or transfers tyrosine to tRNATyr. This indicates that the structural division of TyrTS is equally a functional one: the N-terminal structural domain catalyses tyrosine activation while the disordered C-terminal domain carries major determinants in tRNA binding.

Adenosine Monophosphate↗
PubMed1979

A retrospective analysis of conservative versus active management in severe open myelomeningocele.

88 patients with thoraco-lumbar myelomeningocele not operated upon in the 1976-1977 period were compared with 76 patients with the same condition operated upon in the 1964-1971 period. There was no significant difference in sex and hydrocephalus at birth in the two groups. Mortality was less in the surgically treated group only after the age of three months. Ventriculitis appeared to be related to the incidence of hydrocephalus in the surgically treated group. There was a reduced risk of developing progressive hydrocephalus after birth in the untreated group, and the neurological status of the survivors at one year was the same in both groups. Interpreting the results as indicating that early surgery increased the incidence of progressive hydrocephalus and ventriculitis, and might increase survival of more disabled infants, then non-surgical treatment is justified since survivors are no worse as a result of this non-active approach.

Cerebral Ventricles↗
PubMedDate not supplied

Engineering of tyrosyl tRNA synthetase.

The gene encoding the enzyme tyrosyl tRNA synthetase from Bacillus stearothermophilus has been systematically altered using synthetic oligonucleotides as mutagens. The construction of mutations has been facilitated by using strains of bacteria defective in mismatch repair and also by utilising a genetic marker in the M13 strain (such as an amber mutation, or an EcoK or EcoB site) which allows selection for the progeny of M13 replication derived from the minus (mutagenized) strand. Several mutations have been constructed in the ATP binding site to elucidate the roles of individual residues in catalysis and substrate binding and it has even been possible to construct mutants which have improved affinity for ATP. Mutations in various surface lysine and arginine residues have allowed us to identify potential contacts with the tRNA, and indicate that a cluster of basic residues close to the C-terminus of the enzyme probably makes important interactions with the tRNA.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

A large increase in enzyme-substrate affinity by protein engineering.

A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme-substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 leads to Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 leads to Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

Hydrogen bonding and biological specificity analysed by protein engineering.

The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5-1.5 kcal mol-1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further approximately 3 kcal mol-1.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

The solution structure of human epidermal growth factor.

The epidermal growth factors (EGFs) are powerful mitogens for a wide variety of cells in culture; human EGF (hEGF), known as urogastrone, also inhibits gastric acid secretion in vivo. The transforming growth factors (TGF-alpha) are related to the EGF family both in sequence and activity and EGF-like sequences are often observed in a wide range of functionally unrelated proteins. Attempts to examine the structure of EGF by diffraction methods have not yet succeeded because of difficulties with crystallization. We report here a three-dimensional structure of a biologically active derivative (residues 1-48) of the 53-residue human EGF. An analysis of high resolution 1H nuclear magnetic resonance (NMR) spectra was used together with a combination of distance geometry, restrained energy minimization and restrained molecular dynamics methods. The three-dimensional structure provides a basis for understanding the properties of EGFs and for predicting the structures of homologous sequences in other proteins.

Chemical Phenomena↗
PubMedDate not supplied

Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I-target for antitumor drugs.

The gene encoding human DNA topoisomerase (topo) I, the target of numerous anticancer drugs, has been subcloned into bacterial, yeast and baculovirus-based expression systems in attempts to overexpress the enzyme for extensive structural and functional characterisation. Expression in E.coli produced a protein which was not suitable for structural studies. Expression in the yeast system was more successful enabling the enzyme to be purified and characterised. However, the resulting yield was modest for our requirements and the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that topo I from human placental tissue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fragment and shown by N-terminal sequence analysis that it starts at Lysine-191. This information was used to construct vectors which direct the overexpression of this fragment in baculovirus infected insect cells. The recombinant protein has been purified to homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and retains all of the DNA driving activities of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides and conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichroism technique has been further developed and applied for real-time monitoring of supercoiled (sc) DNA relaxation by the enzyme and for comparative analysis of inhibition of 68kDa topo I by camptothecin (CPT).

Animals↗
PubMedDate not supplied

Walking speed in children and young adults with neuromuscular disease: comparison between two assessment methods.

Self-selected walking speed is being increasingly used as a primary outcome measure in the management of neuromuscular disease. It would be useful if the speed recorded in the gait laboratory represented the child's walking speed in the community. This study investigated the difference in self-selected walking speeds between a 10-meter walk, as measured during instrumented gait analysis, and a 10-minute walk. The authors found that self-selected walking speed during the 10-minute walk was slower than the self-selected walking speed recorded during the 10-meter walk. The former may be more representative of walking speed in the community setting. Walking speed measured during walks of 10 minutes or more should become an integral part of gait laboratory evaluation.

Adolescent↗