Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.
 ...
PMID:Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group. 0 72

Earlier studies have shown that native phenylalanyl-tRNA synthetase from baker's yeast contains two different kinds of subunits, alpha of molecular weight 73000 and beta of molecular weight 63000. The enzyme is an asymmetric tetramer alpha-2beta-2, which binds two moles of each ligand per mole. Incubation of the purified enzyme with trypsin results in an irreversible conversion: the alpha-subunit remains apparently unchanged but beta is rapidly degraded and yields a lighter species beta of molecular weight 41000. The trypsin-modified enzyme is an alpha-2beta-2 molecule which can still activate phenylalanine but cannot transfer it to tRNA-Phe; furthermore it does not bind tRNA-Phe but its kinetic parameters are identical to those of the native enzyme with respect to ATP and phenylalanine. Therefore the two beta subunits play a critical part in tRNA binding. Isolated alpha or beta subunits exhibit no significant activity and both types of subunit seem to be required for phenylalanine activation.
 ...
PMID:Modification of phenylalanyl-tRNA synthetase from baker's yeast by proteolytic cleavage and properties of the trypsin-modified enzyme. 16 41

Yeast phenylalanyl-tRNA synthetase, an enzyme with an alpha2beta2 structure, has two active sites for phenylalanine, tRNAphe, phenylalanyladenylate and phenylalanyl-tRNAphe. Determination of phenylalanine binding properties to the free enzyme by equilibrium dialysis shows that only one mole of amino acid binds per mole of enzyme, i.e. absolute negative cooperativity. Binding of the amino acid in the presence of tRNA or of ATP and PPi unmasks the second phenylalanine binding site. The difference between the affinities at the tight and loose binding sites under such conditions is about 10--15. Titration of phenylalanyladenylate sites by the burst of ATP consumption shows the formation of a (enzyme-phenylalanyladenylate)2 complex in the presence of pyrophosphatase; however, the two sites differ widely in their affinity as shown by dialysis experiments. Measurements of hydrolysis rates of enzyme-bound phenylalanyladenylate suggests that when only the high-affinity adenylate site is occupied, the other protomer can still bind phenylalanine and ATP (in the presence of phenylalanine). Two moles of Phe-tRNAphe bind to the enzyme with a very high affinity (Kd less than 48 nM). The presence of millimolar concentrations of ATP, phenylalanine and pyrophosphate triggers negative cooperativity and under these conditions only one mole of Phe-tRNAphe is bound per mole of enzyme with a Kd value of 0.15 muM. The present results give support to interprotomer catalytic cooperativity in the mechanism of action of yeast phenylalanyl-tRNA synthetase.
 ...
PMID:Non-equivalence of the sites of yeast phenylalanyl-tRNA synthetase during catalysis. 32 9

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA results in a binding of 1 mole of the reagent per 1 mole of the enzyme. Exhaustive alkylation of phenylalanyl-tRNA synthetase completely blocks the aminoacylation and partially inhibits the reaction of ATP--[32P]pyrophosphate exchange. Removal of the tRNA moiety of the reagent by hydrolysis of the ester bond N-chlorambucilyl-phenylalanine and terminal adenosine does not result in a restoration of ATP--[32P]pyrophosphate exchange and aminoacylation activity. The latter result may testify a chemical modification of amino acid residues essential for enzymatic activity. Possibility of blocking one of the two tRNA binding sites is discussed.
 ...
PMID:[Modification of one tRNA recognition site of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA]. 36

Complexes between tRNAPhe (yeast), tRNASer (yeast) and tRNATyr (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results. 1. Two moles of tRNAPhe can be bound to one mole of phenylalanyl-tRNA synthetase with a binding constant greater than 10(6) M-1. The binding constants for both tRNAs are very similar; the binding sites are independent of each other. Omission of Mg2+ does not prevent binding. 2. Two moles of tRNASer can be bound to one mole of Seryl-tRNA synthetase; the binding of the first and second tRNA is non-equivalent, K1 greater than 10(6) M-1, K2 is determined to be 1.3 X 10(5) M-1 at pH 7.2. Omission of Mg2+ prevents complex formation. 3. Tyrosyl-tRNA synthetase behaves very similarly to seryl-tRNA synthetase. The binding constant for the weakly bound tRNA is 2.3 X 10(5) M-1 at pH 7.2, and 2.5 X 10(6) M-1 at pH 6.0. No complexes are observed in the absence of Mg2+. 4. Unspecific binding was only obtained with phenylalanyl-tRNA synthetase. It binds tRNASer (yeast), tRNAAla (yeast) and tRNATyr (E. coli) with a binding constant about 100 times lower compared to its cognate tRNA. The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non-equivalence of binding sites.
 ...
PMID:Equivalent and non-equivalent binding sites for tRNA on aminoacyl-tRNA synthetases. 110 Mar 84

The effect of modification of carboxylic groups of phenylalanyl-tRNA synthetase by p-toluene sulfonate N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbodiimide (CMEC) on the activity of the enzyme was investigated. It was shown that modification of two moles of carboxylic groups per mole of the enzymes leads to the diminution of negative charge of the enzyme and to inactivation in ATP-[32P]PPi-exchange and aminoacylation reactions. The inactivation is completely reversed by mild alkaline hydrolysis. ATP in concentration 2 X 10(-4) M partially protects the enzyme against inactivation, protective effect being stimulated by Mg2+ and 0.4-0.7 moles of carboxylic groups per mole of the enzyme are protected against inactivation is observed although the depth of modification is increased. Other substrates do not have protective effect. Modification of the enzyme by CMEC increases Kdiss value of [14C]-Phe-tRNA enzyme complex and Km value for tRNAPhe in aminoacylation by factor of three. Vmax for all substrates in both aminoacylation and leads to 40% increase of Hill's coefficient for ATP in ATP-[32P]PPi-exchange reaction but not in aminoacylation. The carboxylic groups modified by CMEC are assumed to take part in ATP recognition and in catalysis of the ATP conversion and in catalysis of transfer of activated amino acid residues on tRNA.
 ...
PMID:[Role of the carboxylic groups in interaction of phenylalanyl-tRNA synthetase with substrates]. 703 45