scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

scholarly journals Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase

1993 ◽  
Vol 291 (3) ◽  
pp. 811-816 ◽  
Author(s):  
D Communi ◽  
K Takazawa ◽  
C Erneux
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Inositol 1,4,5 Trisphosphate

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

Site-directed mutagenesis of farnesyl diphosphate synthase; effect of substitution on the three carboxyl-terminal amino acids

1994 ◽  
Vol 72 (1) ◽  
pp. 75-79 ◽  
Author(s):  
Tanetoshi Koyama ◽  
Kazuhiro Saito ◽  
Kyozo Ogura ◽  
Shusei Obata ◽  
Ayumi Takeshita
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Farnesyl Diphosphate ◽  
Site Directed Mutagenesis ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Directed Mutation ◽  
Catalytic Function ◽  
Terminal Amino

Site-directed mutation was introduced into the gene for the farnesyl diphosphate synthase of Bacillus stearothermophilus. To investigate the significance of the three C-terminal amino acids, where arginine is completely conserved throughout the farnesyl diphosphate synthases of prokaryotes and eukaryotes, three kinds of mutant enzymes, R295V, D296G, and H297L, which have replacements of arginine-295 with valine, aspartate-296 with glycine, and histidine-297 with leucine, respectively, were overproduced and purified to homogeneity. All of the three mutant enzymes showed similar catalytic activities to that of the wild-type enzyme, indicating that the basic amino acids including the conserved arginine in the C-terminal region are not essential for catalytic function. They were also similar to the wild-type enzyme with respect to pH optima, thermostability, reaction product, and kinetic parameters for allylic substrates. However, their Km values for isopentenyl diphosphate are approximately twice that of the wild type.

scholarly journals Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily

1999 ◽  
Vol 344 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Melissa BOWKER-KINLEY ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Catalytic Domain ◽  
Heat Shock Protein 90 ◽  
Enzymic Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
C Terminus ◽  
Km Value

In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent Vmax values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent Km value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 μM). In contrast, Gly-284Ala had only a slightly higher Km value than the wild-type enzyme (28 versus 16 μM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The Kd value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 μM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].

scholarly journals Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans

1994 ◽  
Vol 302 (1) ◽  
pp. 291-295 ◽  
Author(s):  
A Moreau ◽  
M Roberge ◽  
C Manin ◽  
F Shareck ◽  
D Kluepfel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzyme Activity ◽  
Streptomyces Lividans ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Protein Mutation ◽  
Acidic Residues

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

scholarly journals Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase

1991 ◽  
Vol 280 (1) ◽  
pp. 125-129 ◽  
Author(s):  
K Takazawa ◽  
C Erneux
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Protein A ◽  
Restriction Enzymes ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Catalytically Active ◽  
Inactive Protein

In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein [clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem. J. 272, 107-112]. Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes. The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way. The catalytic domain is thus located at the C-terminal end of the protein. A series of 5′ deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids. Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain. Affinity chromatography on CaM-Sepharose of 5′ and 3′ deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

scholarly journals Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

1991 ◽  
Vol 279 (1) ◽  
pp. 35-41 ◽  
Author(s):  
R Chambert ◽  
M F Petit-Glatron
Keyword(s):  
Bacillus Subtilis ◽  
Intermediate Formation ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Chain Elongation ◽  
Directed Mutagenesis ◽  
Water Mixture ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Amino Acid Residue 147 of Human Aldosterone Synthase and 11β-Hydroxylase Plays a Key Role in 11β-Hydroxylation*

2000 ◽  
Vol 85 (3) ◽  
pp. 1261-1266
Author(s):  
Angela Fisher ◽  
Robert Fraser ◽  
John MC Connell ◽  
Eleanor Davies
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Affinity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Active Centers ◽  
Hormone Production ◽  
Aldosterone Synthase ◽  
Linear Distance ◽  
Aldosterone Production

Abstract A number of amino acids differ between aldosterone synthase and 11β-hydroxylase. To assess their importance in determining the different functional specificities, we substituted aldosterone synthase-specific (aspartate D147, isoleucine I248, glutamine Q43, and threonine T493) with 11β-hydroxylase-specific amino acids (glutamate E147, threonine T248, arginine R43, and methionine M493), respectively. I248T, Q43R, and T493M had no effect on steroid production compared to wild-type aldosterone synthase. However, CYP11B2-D147E caused a significant increase in corticosterone production and a smaller increase in aldosterone production from 11-deoxycorticosterone (DOC). This appeared to be predominantly due to an increase in the 11β-hydroxylation of DOC to corticosterone mediated by a decrease in Km, which was 1.4 μmol/L for the mutant compared with 5μ mol/L for the wild-type enzyme. CYP11B2-D147E had no effect on the conversion of 11-deoxycortisol to cortisol. The reverse construct (CYP11B1-E147D), substituting the 11β-hydroxylase residue with the aldosterone synthase equivalent, decreased the conversion of DOC to corticosterone, which was mediated by an increase in Km that was 7.5 μmol/L for the mutant compared with 2.5 μmol/L for the wild-type enzyme. Again, the conversion of 11-deoxycortisol to cortisol was unimpaired. Thus, amino acid 147 is involved in the transformation of the 17-deoxysubstrate, but not the 17α-hydroxysubstrate. The results demonstrate that a conservative change in amino acid, even at some linear distance from known active centers, can significantly affect enzyme substrate affinity and subsequent steroid hormone production.

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