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H Jakubowski

Publications and source records attributed to H Jakubowski.

At least 73 records · Page 4Linked to original sources
PubMed1980

Conformational changes during enzyme catalysis: role of water in the transition state.

The entropy of activation for the synthesis of Ile-tRNA is high and positive. The only likely source of a high delta S is the loss of structured water as the enzyme . substrate complex moves toward the transition state. This requires a change in the orientation or nature of water-organizing residues in the interface between the enzyme . substrate complex and the water. Such changes, which may be some distance from the "active site," are coupled to the active site in such a way that the increased entropy and decreased free energy of the water--enzyme interface is available at the "active site" to reduce the free energy of activation. The effects of Hofmeister anions on KmS and KcatS are consistent with the entropy data.

Amino Acyl-tRNA Synthetases↗
PubMed1980

The plant aminoacyl-tRNA synthetases. 2'-DeoxyATP and ATP in reactions catalysed by yellow lupin aminoacyl-tRNA synthetases.

2'-Deoxyadenosine triphosphate (dATP) is not a substrate for valyl-tRNA, leucyl-tRNA and isoleucyl-tRNA synthetases from yellow lupin seeds. Yellow lupin seryl-tRNA, phenylalanyl-tRNA, tyrosyl-tRNA, arginyl-tRNA, lysyl-tRNA and methionyl-tRNA synthetases use dATP as a substrate both in aminoacyl deoxyadenylate formation and tRNA aminoacylation reactions. Generally, dATP is a poorer substrate, being 3 - 50% as effective as ATP. None of the other nucleoside triphosphates tested was shown to be a substrate for these enzymes. Specificity (k cat/Km) towards ATP is greater in tRNA aminoacylation than in pyrophosphate exchange reaction catalysed by seryl-tRNA synthetase. Energy of activation is the same (Ea = 18.5 kcal) for dATP- and ATP-dependent pyrophosphate exchange. Both dATP- and ATP-dependent tRNA aminoacylation reactions exhibit the same temperature dependence with Ea = 9.0 kcal. Half lives of the enzyme-bound seryl adenylate and deoxyadenylate are 2 min and 4 min, respectively (pH 8.0, 25 degrees C). Both enzyme-bound phenylalanyl adenylate and deoxyadenylate exhibit the same stability (half lives 0.3 min), and enzyme-bound tyrosyl deoxyadenylate is hydrolysed faster (half life 0.6 min) than tyrosyl adenylate (half life 2.5 min). Serine is transferred both from enzyme-bound seryl adenylate and deoxyadenylate to tRNA with the same 60% efficiency. One mol of ATP or 1.1 mol of dATP is hydrolysed per one mol of seryl-tRNA formed during aminoacylation in the complete ATP- or dATP-dependent system, respectively. AMP concentrations (up to 0.5 mM), which do not affect the equilibrium of the ATP-dependent tRNA esterification with serine, significantly change the equilibrium of the dATP-dependent reaction.

Adenosine Triphosphate↗
PubMed1978

Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate.

As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.

Adenosine Monophosphate↗
PubMed1978

The mechanism of the aminoacylation of transfer ribonucleic acid. The kinetics and stoichiometry of the lysis of aminoacyl-tRNA.

It is often stated that the aminoacylation of transfer RNA proceeds in discrete steps: (formula: see article). If this is a complete description of the reaction, the reverse overall formation of ATP should not be more rapid than the formation of Enz . (AA approximately AMP). We show for four different amino acid:tRNA ligases that lysis of AA-tRNA (with PPi and AMP) to ATP is faster than lysis of AA-tRNA (with AMP only) to Enz . (AA approximately AMP). This requires that the transition state proceeds from a quaternary complex of PPi, AMP, AA-tRNA and Enz. From the law of microscopic reversibility, this requires that in the forward reaction the AA-tRNA bond be formed before PPi leaves the enzyme complex. Therefore, the forward reaction passes through the quaternary complex Enz . ATP . AA . tRNA. (In view of recent evidence of the specific requirement of two cations, the complex is accurately described as senary).

Adenosine Monophosphate↗
PubMed1978

Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B.

The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation.

Amino Acyl-tRNA Synthetases↗
PubMed1977

The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.

The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Fractionation of plant aminoacyl-tRNA synthetases on tRNA-Sepharose columns.

It has been shown that tRNA-Sepharose, a chromatographic adsorbent containing unfractionated tRNA bound to a Sepharose matrix, is a useful, group-specific adsorbent for fractionation of the plant aminoacyl-tRNA synthetases. Conditions are described in which Val-, Trp-, Phe-, Leu- and Ile-tRNA synthetases from yellow lupin seeds can be separated from each other on the tRNA-Sepharose columns. Factors affecting affinity chromatography on the t-RNA-Sepharose columns are discussed. The affinity chromatography procedure for the purification of lupin Ser-tRNA synthetase to homogenity is described.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Transfer RNA methyltransferases from yellow lupin seeds: purification and properties.

tRNA methyltransferases from extract of yellow lupin seeds were purified over 300-fold by the methods based on hydrophobic and affinity chromatography. However, in the most active fractions the methylating enzymes were over 2000 purified. The purified enzyme fractions catalysed the formation of 1-methyladenine and 5-methylcytosine using E. coli B and B. subtilis tRNAs as substrates and S-adenosylmethionine as the methyl donor. They were unable to methylate their own endogenous tRNA but they were capable of methylating tRNA of some other lupinus species. Whereas the patterns of methylated constituents of tRNA of some other lupinus and B. subtilis were quite similar, they differed considerably from those obtained with lupin species tRNAs. Some properties of purified methyltransferases from yellow lupin seeds have been described.

Adenine↗