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At least 1,063 records · Page 59Linked to original sources
PubMed2000

The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5.

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b5, three mutants at a highly conserved residue Val45, which is a member of heme hydrophobic pocket residues have been characterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were constructed to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyrosine still sitting inside the hydrophobic pocket, while the side chain of the mutant V45E and V45H shift away from the heme pocket. The redox potentials of mutants V45Y, V45H, V45E and wild-type of cytochrome b5 are -35 mV, 8 mV, -26 mV and -3 mV, respectively. The bigger change of the V45Y on redox potential is due to the close contact between the hydroxyl group and the heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stability of these mutants towards heat have been observed with the order: WT > V45Y-V45H > V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT > V45Y > V45H > V45E. These results suggest that the difference in the hydrophobic free energy is a major factor contributing to the stability of the Val45 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Val45 plays an important role in the stability and redox potential of the protein.

Acids↗
PubMed1993

Molecular defect in factor IX Tokyo: substitution of valine-182 by alanine at position P2' in the second cleavage site by factor XIa resulting in impaired activation.

Utilizing polymerase chain reaction and directly sequencing the amplified exon 6 of the factor IX gene derived from a mild hemophilia Bm patient, we have identified a T to C mutation at nucleotide 20,525. This point mutation predicted a Val182 to Ala substitution in the abnormal factor IX molecule, designated as factor IX Tokyo. The patient manifested a low factor IX activity and a moderately prolonged ox-brain prothrombin time but a normal factor IX antigen level in plasma. Immunopurified factor IX derived from the patient was found to have a normal molecular weight but a reduced specific activity (23% of normal). Limited proteolysis by activated factor XI or by a snake venom-derived factor X-activating enzyme was considerably delayed, indicating the presence of structural alteration(s) most probably at or near the second enzyme-cleavage site. Once activated, however, factor IXa Tokyo was able to activate factor X normally and was inactivated by antithrombin III also in a normal fashion. The structural model of factor IXa and a docking model of factor IX and activated factor VII (factor VIIa) suggested that the Val182 to Ala substitution would not affect the local conformation of the catalytic domain. This mutation would rather loosen the fitness of the molecule into the substrate-binding pocket of factor VIIa due to a shorter side chain of the Ala substitution at the P2' position of the second cleavage site.

Adolescent↗
PubMed1994

Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B.

The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (Val10Arg cystatin C), the inhibition constant, K(i), increased 31-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin B. When the wild-type form of the inhibitor was used, the corresponding effect on K(i) was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-171, no such difference in how K(i) is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on K(i) indicate that the additional positive charge introduced in Val10Arg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A K(i) value of 0.13 nM was obtained for the interaction of Val10Arg cystatin C with papain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study.

Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.

Base Sequence↗
PubMed1991

Thermodynamics and mechanism of alpha helix initiation in alanine and valine peptides.

We used molecular dynamics simulations to study the folding/unfolding of one of turn of an alpha helix in Ac-(Ala)3-NHMe and Ac-(Val)3-NHMe. Using specialized sampling techniques, we computed free energy surfaces as functions of a conformational coordinate that corresponds to alpha helices at small values and to extended conformations at large values. Analysis of the peptide conformations populated during the simulations showed that alpha helices, reverse turns, and extended conformations correspond to minima on the free energy surfaces of both peptides. The free energy difference between alpha helix and extended conformations, determined from the equilibrium constants for helix unfolding, is approximately -1 kcal/mol for Ac-(Ala)3-NHMe and -5 kcal/mol for Ac-(Val)3-NHMe. The mechanism observed in our simulations, which includes reverse turns as important intermediates along the helix folding/unfolding pathway, is consistent with a mechanism proposed previously. Our results predict that both peptides (but especially the Ala peptide) have a much larger equilibrium constant for helix initiation than is predicted by the helix-coil transition theory with the host-guest parameters. We also predict a much greater difference in the equilibrium constants than the theory predicts. Insofar as helix initiation is concerned, our results suggest that the large difference between the helical propensities of Ala and Val cannot be explained by simple concepts such as side-chain rotamer restriction or unfavorable steric interactions. Rather, the origin of the difference appears to be quite complicated because it involves subtle differences in the solvation of the two peptides. The two peptides have similar turn-extended equilibria but very different helix-turn equilibria, and the difference in helical propensities reflects the fact that the helix-turn equilibrium strongly favors the turns in Ac-(Val)3-NHMe, while it favors the helices in Ac-(Ala)3-NHMe. We also computed thermodynamic decompositions of the free energy surfaces, and these revealed that the helix-turn equilibria are vastly different primarily because the changes in peptide-water interactions that accompany helix-to-turn conformational changes are qualitatively different for the two peptides.

Alanine↗
PubMed1989

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity.

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.

Amino Acid Sequence↗
PubMed1989

Mutation of glycine 49 to valine in the alpha subunit of GS results in the constitutive elevation of cyclic AMP synthesis.

The G-protein GS couples hormone-activated receptors with adenylyl cyclase and stimulates increased cyclic AMP synthesis. Transient expression in COS-1 cells of cDNAs coding for the GS alpha-subunit (alpha S) or alpha S cDNAs having single amino acid mutations Gly49----Val or Gly225----Thr elevated cyclic AMP levels, resulting in the activation of cyclic AMP dependent protein kinase. Stable expression in Chinese hamster ovary cells of alpha S Val49 cDNA resulted in a small constitutive elevation of cyclic AMP that was sufficient to persistently activate cyclic AMP dependent protein kinase activity 1.5-2-fold over basal activity. Stable expression of wild-type alpha S or alpha S Thr225 in Chinese hamster ovary cells was less effective in sustaining elevated cyclic AMP synthesis and kinase activation compared to alpha SVal49.

Animals↗
PubMed1975

Purification of human placenta phenylalanine, valine, methionine, glucine, and serine transfer ribonucleic acids.

By using column chromatography on varied media, the purification of several individual tRNAs from human placenta has been achieved. The crude human placenta tRNA was isolated using phenol extraction at pH 4.5 followed by DEAE-cellulose chromatography (B. Roe (1975), Nucleic Acids Res. 2, 21-42) and initially fractionated on BD-cellulose at neutral pH. Subsequent chromatography of the partially purified tRNA using high-speed, high-pressure liquid chromatography on RPC-5 and Aminex A-28 coupled with chromatography on BD-cellulose at acidic pH and on DEAE-Sephadex A-50 significantly shortened isolation time for milligram quantities of several pure tRNA species. Those tRNAs from human placenta obtained in a purity greater than 1.2 nmol/A260 unit are tRNAPhe, tRNAMet(i), tRNAVal(1a), tRNAVal(1b), and tRNAGly(1), while those obtained at purity of at least 0.8 nmol/A260 unit are tRNASer2 and tRNASer3. In addition, the use of Aminex A-28 as a chromatographic system for the isolation of tRNA is discussed.

Chromatography, High Pressure Liquid↗