scholarly journals The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

1996 ◽  
Vol 318 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Fumio SAKANE ◽  
Masahiro KAI ◽  
Ikuo WADA ◽  
Shin-ichi IMAI ◽  
Hideo KANOH
Keyword(s):  
Enzyme Activity ◽  
Binding Sites ◽  
Enzyme Activities ◽  
Catalytic Domain ◽  
Zinc Fingers ◽  
Diacylglycerol Kinase ◽  
Site Directed Mutagenesis ◽  
Atp Binding ◽  
Wild Type

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

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.

scholarly journals Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

2002 ◽  
Vol 13 (4) ◽  
pp. 1190-1202 ◽  
Author(s):  
Hélène Defacque ◽  
Evelyne Bos ◽  
Boyan Garvalov ◽  
Cécile Barret ◽  
Christian Roy ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Membrane Surface ◽  
Latex Bead ◽  
Actin Assembly ◽  
Wild Type ◽  
Membrane Surfaces ◽  
Organelle Membrane

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process ( Defacque et al., 2000b ). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

scholarly journals Difference in Stability of the N-domain Underlies Distinct Intracellular Properties of the E1064A and H1069Q Mutants of Copper-transporting ATPase ATP7B

2011 ◽  
Vol 286 (18) ◽  
pp. 16355-16362 ◽  
Author(s):  
Oleg Y. Dmitriev ◽  
Ashima Bhattacharjee ◽  
Sergiy Nokhrin ◽  
Eva-Maria E. Uhlemann ◽  
Svetlana Lutsenko
Keyword(s):  
Phenotypic Variability ◽  
Copper Metabolism ◽  
Complete Loss ◽  
Atp Binding ◽  
Wild Type ◽  
Helical Hairpin ◽  
The Difference ◽  
The Stability ◽  
Golgi Network

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu1064 and His1069 is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.

In vitro Mutagenesis of Human Dihydropteridine Reductase at the Active Site and at Altered Sites Found in the Reductases of Deficient Children

Pteridines ◽  
1996 ◽  
Vol 7 (4) ◽  
pp. 123-136 ◽  
Author(s):  
Hong-Ping Zhang ◽  
Nan Yang ◽  
Wilfred L. F. Armarego
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
General Procedure ◽  
Maximum Velocity ◽  
Order Rate Constant ◽  
Site Directed Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Dihydropteridine Reductase ◽  
Proton Source

Summary A general procedure for in vitro site-directed mutagenesis of the wild-type dihydropteridine reductase gene has been used successfully to make eight mutant proteins. Five mutations were at the active site, viz Tyrl50His, Tyrl50Ser, Tyrl50Phe, Tyr150Glu and Tyrl50Lys. The proteins were expressed as glutathione S-transferase fusion proteins from which the unconjugated reductases were obtained by thrombin cleavage. The kinetic parameters of the conjugated and unconjugated reductases were measured using natural quinonoid R-7,8(6H)-dihydrobiopterin and non-natural quinonoid RS-6-methyl-7,8(6H)-dihydropterin and NADH. The kcat (maximum velocity at saturating concentrations of substrates) and kcatl Km (first order rate constant at low concentration of substrates) values show that the phenolic OH of Tyr 150 was the most likely proton source to complete the hydride reduction of the quinonoid pterin cofactor. However in the absence of a proton source at residue 150, measurable enzyme activities were observed indicating that a proton was relayed via a water molecule(s) from some neighbouring acidic amino acid residue. Three mutant dihydropteridine reductases, which were found in defective children, have been similarly attempted, viz GlylSlSer, Gly23Asp and a threonine insertion at position 123. The enzyme activities of the first two mutant reductases were consistent with the severity of the disease. The unconjugated reductase from the third mutation could not be obtained due to proteolysis but the fusion protein was enzymically active.

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 TET-mediated 5-methylcytosine oxidation in tRNA promotes translation

2020 ◽  
pp. jbc.RA120.014226
Author(s):  
Hui Shen ◽  
Robert Jordan Ontiveros ◽  
Michael C Owens ◽  
Monica Yun Liu ◽  
Uday Ghanty ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Biological Function ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Wild Type ◽  
Oxidation Activity ◽  
Tet Enzymes ◽  
Mediated Oxidation

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

scholarly journals PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. C528-C534 ◽  
Author(s):  
Shiuhyang Kuo ◽  
Ann L. Chokas ◽  
Richard J. Rogers ◽  
Harry S. Nick
Keyword(s):  
Binding Sites ◽  
Manganese Superoxide Dismutase ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Site Directed Mutagenesis ◽  
Cellular Respiration ◽  
Point Analysis ◽  
Specific Regulation

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

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