Key active sites of proteases and protease inhibitors: a review / 生物工程学报

Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.

In-silico analysis of Porcine and recombinant Insulin activity on Glycogen

The study focuses on the anti-diabetic activity by molecular simulation of Recombinant Insulin, PorcineInsulin, and Glycogen. The sequence of these three molecules was retrieved, and 3D structures weremodeled. A total of two different molecular simulations were carried out. The simulations were done usingAutodock software. Initially, the downloaded PDB structures were docked with glycogen and the secondbetween the active site peptide models of both insulin molecules based on castP prediction with glycogenmolecule. The results were analyzed by Ramachandran plot for model prediction, and the binding energywas set as criteria to determine the best-docked model. The binding energy of recombinant insulin, porcineinsulin with glycogen was 0.32 and -1.09 respectively. Similarly, the binding energy for peptide modelswith glycogen molecule was found to be +1.09 and +6.76 respectively. Based on the results, it wasconcluded that the recombinant insulin has higher affinity than the porcine insulin.

Research progress in structure and function of pectin methylesterase / 生物工程学报

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.

Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim:To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryoticMSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme.Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.Place and Duration of Study: School of Biotechnology, MaduraiKamaraj University, Madurai, India, between 2007-2013. Methodology:Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Results:Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the templatebinding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9thposition from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regionsare highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, inthe eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6thposition are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes.Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present

Effect of Inula cappa on CYP450 Enzyme in Rats by Cocktail Probe Drugs / 中国实验方剂学杂志

Objective:To study on the effect of Inula cappa extract on the activities of six cytochrome P450(CYP450) enzymes in rats by Cocktail probe method. Method:Rats in the I. cappa high and low dose groups were given oral administration of active fractions of I. cappa at a dose of 8.127,2.709 g·kg-1·d-1 of the material for 7,14 d,repectively.Probe drugs(caffeine,midazolam,tolbutamide,omeprazole,metoprolol,chlorzoxazone) were simultaneously injected to rats after administration.Plasma was collected at different time after the administration of probe drugs.The plasma concentrations of these six probes were measured by UPLC-MS and their corresponding pharmacokinetic parameters were calculated with DAS 2.0. Result:Compared with the control group,only the apparent volume of distribution(V) of midazolam was increased;area under the curve(AUC0-t and AUC0-∞)and half-life period(T1/2) of caffeine,midazolam,tolbutamide and omeprazole were increased and the clearance rate(CL) of them was decreased in rats of I. cappa groups.But there were no differences in pharmacokinetic parameters of chlorzoxazone and metoprolol. Conclusion:I. cappa can reduce the enzymatic activities of CYP3A,CYP1A2,CYP2C9 and CYP2C19 in rats at different degree,among which CYP3A is the strongest.

Study on effect of different extraction methods on anti-inflammatory and analgesic activity of Anemone hupehensis / 重庆医学

Objective To investigate the effect of different extraction methods on anti-inflammatory and analgesic activity of Anemone hupehensis.Methods The different abstracts were prepared from the whole herb of Anemone hupehensis.The analgesic effect was observed by adopting the mouse torsion and electric heating plate method,and the anti-inflammatory activity was comprehensively evaluated by using the mouse ear tumefaction,toe tumefaction and tampon granulation tumefaction exprements.Results Compared with the blank model group,the anti-inflammatory action difference of low dose in the water layer parts of mouse ear tumefaction,toe tumefaction and tampon granulation tumefaction had no statistical significance(P＞0.05),and the extracting parts of rest doses all had significant anti-inflammatory and analgesic effect (P＜0.05).Ethyl acetate part had strongest activity in the electric heating plate experiment.N-butanol part had strongest activity in the ear tumefaction,toe tumefaction,tampon granulation tumefaction experiments and torsion method.Conclusion The whole herb of Anemone hupehensis has prominent anti-inflammatory and analgesic effect,and the ethyl acetate part E and N-butanol part are main effective parts.

Identificación in silico de moléculas potencialmente inhibidoras de CDK5, proteína relacionada con la enfermedad de Alzheimer / Identification in silico of potentially inhibitive molecules of CDK5, protein related with the Alzheimer´s disease

Introducción: La enfermedad de Alzheimer exhibe un compromiso neurodegenerativo e irreversible. Hoy, numerosas investigaciones promueven la inhibición de algunas quinasas para su tratamiento, de especial mención la CDK5. Objetivo: Identificación de nuevas moléculas con posibilidad de interactuar con la proteína quinasa dependiente de ciclina 5, CDK5, inhibiendo su función. Material y Métodos: Se realizó un estudio in silico, para lo cual se extrajeron 911 moléculas de pubchem, y mediante AutoDock Vina se hicieron acoplamientos moleculares con la proteína CDK5 extraída de Protein Data Bank y con un inhibidor conocido para la proteína. Además se realizó un acoplamiento inverso para la identificación de otros posibles blancos moleculares con los mejores ligandos seleccionados. Resultados: Con los resultados obtenidos fueron identificadas cinco moléculas con valores de afinidad entre -11,6 hasta -17,7 Kcal/mol que se unen en el sitio activo de la proteína, de igual forma que lo hace el inhibidor conocido de la misma, e interactúan con los residuos cisteína 83 y glutamina 81. Conclusiones: Las moléculas identificadas pueden interactuar con la CDK5 a nivel de su sitio activo, por lo que podrían actuar como inhibidores de esta quinasa. Esto abre una futura ventana terapéutica en el tratamiento de la enfermedad de Alzheimer)AU)

Introduction: The illness of Alzheimer exhibits a neurodegenerative and irreversible commitment. Today, numerous investigations promote the inhibition of some kinases to the treatment, of special mention the CDK5. Objective: Identification of new molecules witch are able to interact with the cicline dependent kinase protein 5, CDK5, inhibiting their function. Material and Methods: it was carried out a study in silico, for that 911 pubchem molecules were extracted, and by means of AutoDock Vina molecular joining were made with the protein CDK5 extracted from the Protein Data Bank and with a well-known inhibitor for the protein. It was also carried out an inverse joining for the identification of other possible molecular targets with the best selected ligands. Results: With the obtained results five molecules were identified with values of likeness among -11,6 until -17,7 Kcal/mol that joins in the active site of the protein, in the same form that makes it the well-known inhibitor of the CDK5, and interact with the residuals cysteine 83 and glutamine 81. Conclusions: The identified molecules can interact with the CDK5 at level of their active place, for what you/they could act as inhibitors of this quinasa. This opens a future therapeutic window in the treatment of the illness of Alzheimer(AU)

Study on exosite inhibitor of cathepsin K from Herba Ecliptae fractions / 药学学报

Cathepsin K (CTSK) is considered a critical pharmaceutical target in the treatment of osteoporosis. CTSK exerts proteolytic activities against regulatory proteins besides its collagenase function, which may account for some of the adverse reactions when blocked by active site-directed inhibitors. Exosite inhibitors that can discriminate between the therapeutic collagenase and other biological activities of CTSK specifically inhibit the collagenase activity of CTSK without interfering with the other proteolytic activities of the protease. Active recombinant CTSK was expressed in Pichia pastoris, and purified by n-butyl sepharose and SP sepharose column chromatography. Herba Ecliptae is a common traditional Chinese medicine in the treatment of bone diseases. Collagenase assay and benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) substrate assay based on CTSK are applied to verify the exosite inhibitors. n-Butanol extract of Herba Ecliptae are the most active fraction and eclalbasaponin IX isolated from n-butanol fraction is the potential exosite inhibitor of CTSK.

Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis / 生物工程学报

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Screening of Gongshisong's Active Sites for Anti-sports Fatigue / 中国药房

OBJECTIVE:To screen Gongshisong's active sites for anti-sports fatigue. METHODS:Gongshisong extract was prepared with 80% ethanol extraction technology,and extracted with petroleum ether,chloroform,ethyl acetate and n-butyl alco-hol after dispersed with water to obtain the extract. 70 mice were randomly divided into blank control group(1% sodium carboxy-methylcellulose,CMC-Na),positive control group [Rhodiola wallichiana capsules,590 mg/(kg·d)],petroleum ether,chloro-form,ethyl acetate and n-butyl alcohol extracts and aqueous layer of Gongshisong groups(TS,TL,TY,TZ,TW groups). Gong-shisong extracts groups was given relevant medicine 2.5 g(crude drug)/(kg·d),ig,for consecutive 7 days. Exhaustion time of bur-den swimming test was detected. 70 mice were grouped according to above method,and the contents of liver glycogen,muscle gly-cogen and the coefficient of liver were tested in mice. 80 mice were grouped according to above method,and model group was es-tablished additionally(1% CMC-Na). The contents of lactic acid(LA),creatine kinase(CK)and urea nitrogen(BUN)in serum of mice were determined after 90 minutes of unburden swimming. RESULTS:Compared with blank control group,exhaustion time of burden swimming mice in TS,TY and TZ groups prolonged;the content of liver glycogen increased in TY,TZ and TW groups;the content of muscle glycogen increased in TS and TW groups;the contents of BUN,LA and CK in mice increased in model group (P<0.05 or P<0.01). Compared with model group,the serum content of BUN in mice decreased in TS and TY groups;that of LA in mice decreased in TZ and TW groups;that of CK in mice decreased in TS group (P<0.05 or P<0.01). CONCLUSIONS:The petroleum ether and n-butanol extract site and water layer of Gongshisong are good anti-fatigue active sites.

Screening and Fingerprint Analysis of Active Site to Reduce Blood Lipid from Xin-Mai Capsule / 世界科学技术-中医药现代化

This study was aimed to screen main active site to reduce blood lipid from Xin-Mai (XM) capsule and establish HPLC fingerprint of the site, in order to study the correlativity between active site and relevant fractions of its herbs. Solvent extraction was used to separate XM capsule into different polar fractions. Intraperitoneal injection of 75% egg-yolk emulsion was used to establish mice hyperlipidemia models. And the active site was screened. Chromatographic fingerprints of the site and relevant fractions of its herbs were configured by HPLC analysis. The retention time of peaks was utilized as index to evaluate the correlativity. The results showed that lipid-lowering effect of ethyl acetate extract and garlic essential oil was significant (P<0.01). Fingerprint of the active site in XM capsule was established with 28 fingerprint peaks and the assignment results of 27 peaks were indicated. It was concluded that the active sites to reduce blood lipid of XM capsule were ethyl acetate extract and garlic essential oil. The established fingerprint method can effectively determine the correlativity between the active site and its relevant fractions, which contributed to pharmacodynamic material foundation and quality standard.

DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism.

To analyze the active sites of various prokaryotic and eukaryotic DNA polymerases and propose a plausible mechanism of action for the polymerases with the Escherichia coli DNA polymerase I as a model system. Study Design: Bioinformatics, Biochemical and X-ray crystallographic data were analyzed. Place and Duration of Study: Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India. From 2007 to 2012. Methodology: The advanced version of T-COFFEE was used to analyze both prokaryotic and eukaryotic DNA polymerase sequences. Along with this bioinformatics data, X-ray crystallographic and biochemical data were used to confirm the possible amino acids in the active sites of different types of polymerases from various sources. Results: Multiple sequence analyses of various polymerases from different sources show only a few highly conserved motifs among these enzymes except eukaryotic epsilon polymerases where a large number of highly conserved sequences are found. Possible catalytic/active site regions in all these polymerases show a highly conserved catalytic amino acid K/R and the YG/A pair. A distance conservation is also observed between the active sites. Furthermore, two highly conserved Ds and DXD motifs are also observed. Conclusion: The highly conserved amino acid K/R acts as the proton abstractor in catalysis and the YG/A pair acts as a “steric gate” in selection of only dNTPS for polymerization reactions. The two highly conserved Ds act as the “charge shielder” of dNTPs and orient the alpha phosphate of incoming dNTPs to the 3’-OH end of the growing primer.

High-throughput virtual screening for novel dhfr inhibitors of P. Jiroveci from the zinc database.

Many of the opportunistic infections that occur at this late stage can be fatal and since that Pneumocystis carinii pneumonia (PCP) is a leading opportunistic infection found among immunocompromised (CD4 cell < 200) patients worldwide. DHFR is responsible for the growth and maturation of sporozoites stage (life cycle) in Pneumocystis as reported. Currently, 13 million chemical compounds are available for virtual screening in ZINC database. The biological information of four known drug molecules like TMP/SMX, Dapsone, Atovaquone and Pentamidine were collected from the PubChem compound database. Q-Site Finder online tool was used to determine the active site of DHFR in P. jiroveci. LogP values of chemical compounds were identified with the Atom-additive method. Since, existing drugs are synthetic chemicals that give more side effects in Pneumocystis affected patients. Polar surface area value of oxamide (86.18) was predicted to be in the ranges of existing drug values. Pentamidine was proved to be a more efficient ligand based on the dock score of -26.3398 still could not be considered as the natural compound oxamide also was highly comparable with the value of -20.3173. The binding affinity of the selected molecule was analyzed through Pose View and LigPlot.

Comparative protein modelling of metallothionein 1B protein acting as a potential chemoprotector against hepatic neoplasia.

Background and objectives: Primary liver cancer also called hepatocellular carcinoma or hepatoma is the fifth most common cancer in the world with a poor prognosis. An Indian medicinal plant Acanthus ilicifolius shows encouraging results in preventing liver cancer cells from progressing. The aqueous leaf extract (ALE) of the plant was substantially effective in preventing hepatic DNA alterations and sister-chromatid exchanges (a type of chromosomal damage) in tumor-bearing mice. The ALE treatment was able to limit liver metallothionein 1B expression, a potential marker for cell proliferation, and lengthen the mean survival of animals to a significant extent. The findings suggest that Acanthus ilicifolius may be used as a potential chemoprotector against hepatic neoplasia. Methods: The protein ID from Swissprot data base was selected for obtaining the description and function of the protein, its domains structure, post-translational modifications etc. BLAST analysis was performed to identify template protein sequence for metallothionein protein. The comparative modelling of the Metallothionein 1Bby different algorithms like Hidden Markov Model of homology modelling and multiple threading alignments and iterative alignments methods for Ab initio was performed by using the programs like Swiss Model, CPH Model, Wurst and I.TASSES respectively. Results: The obtained models were verified with structure validation programs like, PROCHEK & SAVS. Interpretation and Conclusions: Schrodinger was used for energy refinement of the model. Active site determination through CASTp and surface visualization by Molegro Virtual Docker suggests that this protein can act as a potential drug target.

Construction of the pharmacophore model of acetylcholinesterase inhibitor / 药学学报

Based on ninety three acetylcholinesterase inhibitors (AChEIs) which have the same mechanism of action but are different in structural characteristics, the pharmacophore model for acetylcholinesterase inhibitor was constructed by the CATALYST system. The optimal pharmacophore model with three hydrophobic units, a ring aromatic unit and a hydrogen-bond acceptor unit were confirmed (Weight=3.29, RMS=0.53, total cost-null cost=62.75, Correl=0.93, Config=19.05). This pharmacophore model will act on the double active site of acetylcholinesterase and is able to predict the activity of known acetylcholinesterase inhibitors that are used for clinical treatment of Alzheimer's disease (AD), and can be further used to identify structurally diverse compounds that have higher activity treating with Alzheimer's disease (AD) by virtual screening.

Antibacterial Activity and Antibacterial Action Sites of Bovine Lactoferricin / 微生物学通报

Bovine Lactoferricin is a fragment of polypeptide which derives from N-terminal of bovine lactoferrin when it is digested by pepsin in acid condition. It has many biological functions. This study was designed to research the antibiosis spectrum of LfcinB and the key functional active site of the LfcinB by amino acid substitution and peptide sequence modification. Antimicrobial spectrum of the artificial synthesized LfcinB was determined by agar-well diffusion method. The antibacterial active sites were confirmed by minimal inhibitory concentration assays. After the Cysteine at the third site and the tryptophan at the eighth site of LfcinB were substituted by alanine, or two cysteine of LfcinB were respectively, the minimal inhibitory concentration of the three artificially modified LfcinBs was assayed. Results showed that LfcinB had a broad-spectrum of antibiosis, it could restrain various bacterials, such as Gram-positive bacteria, Gram-negative bacteria, fungus and mycetes. LfcinB was stable to heat and pH, it could not be inactivated by many protease. The tryptophan at the eighth site and the intramolecular disulfide bond formed between two cysteins played a key role for antibiosis, as the functional active sites of LfcinB.

Source and target enzyme signature in serine protease inhibitor active site sequences.

Amino acid sequences of proteinaceous proteinase inhibitors have been extensively analysed for deriving information regarding the molecular evolution and functional relationship of these proteins. These sequences have been grouped into several well defined families. It was found that the phylogeny constructed with the sequences corresponding to the exposed loop responsible for inhibition has several branches that resemble those obtained from comparisons using the entire sequence. The major branches of the unrooted tree corresponded to the families to which the inhibitors belonged. Further branching is related to the enzyme specificity of the inhibitor. Examination of the active site loop sequences of trypsin inhibitors revealed that here are strong preferences for specific amino acids at different positions of the loop. These preferences are inhibitor class specific. Inhibitors active against more than one enzyme occur within a class and confirm to class specific sequence in their loops. Hence, only a few positions in the loop seem to determine the specificity. The ability to inhibit the same enzyme by inhibitors that belong to different classes appears to be a result of convergent evolution.

Mechanistic studies on carboxypeptidase A from goat pancreas Part II: Evidence for carboxyl group.

Studies with substrate analogues and the pH optimum indicated the involvement of carboxyl group in the active site of goat carboxypeptidase A. Chemical modification of the enzyme with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methoI -p-toluene sulphonate, a carboxyl specific reagent, led to loss of both esterase and peptidase activities. Protection studies showed that this carboxyl group was in the active site and was protected by ßphenylpropionic acid and glycyl-L-tyrosine. Kinetic studies also confirmed the involvement of carboxylic group because the enzyme modification with water soluble carbodiimide was a two step reaction which excluded the possibility of tyrosine or lysine which are known to give a one step reaction with this reagent.

Biophysical investigations on the active site of brain hexokinase.

Replacement of Mg (II), the natural activator of brain hexokinase (EC 2.7.1.1) by paramagnetic Mn (II) without affecting the physiological properties of the enzyme, has rendered brain hexokinase accessible to investigations by magnetic resonance methods. Based on such studies, a site on the enzyme, where Mn (II) binds directly with high affinity has been identified and characterized in detail. Use of β, γ-bidentate Cr (III) ATP as an exchange-inert analogue for Mn (II) ATP has shown that Mn (II) binding directly to the enzyme has no catalytic role but another Mn (II) ion binding simultaneously and independently to the enzyme through the nucleotide bridge participates in enzyme function. However, using this direct binding Mn (II) ion and a covalently bound spin label as paramagnetic probes a beginning has been made in mapping the ligand binding sites of the enzyme. Ultra-violet difference spectroscopy has revealed the presence of at least two glucose 6-phosphate locations on the enzyme one of which presumably is the high affinity regulatory site modulated by substrate glucose. Elution behaviour of the enzyme on a phosphocellulose column suggests that glucose induces a specific phosphate site on the enzyme to which the phosphate bearing regulatory ligands of the enzyme may bind

Alternations of Some Enzymological Characterizations of Site-mutagenized DNA Polymerase ? / 第二军医大学学报

The enzymological characterizations of site-mutagenized rat recombinant DNA polymerase?, RQ182 and RQ183 were studied The phosphocellulose column chromatographies showed that the mutant and the wild DNA polymerases were all eluted by about 0.5 mol/ L KCI, but the denatured DNA-cellulose chromatographies showed that although the wild enzyme was eluted by 0.35 mol/L KCI, RQ182 and RQ183 were eluted by 0.55 and 0.45 mol/L KCI, respectively, indicating that the binding abilities to DNA of the mutant enzymes were increased. Km values for the substrate (dTTP)of the wild enzyme, RQ182 and RQ183 were determined as 38.5, 34.5 and 111.1 ?mol/L, respectively,and the Km values for the primer (oligo(dT)) were 1.28, 1.96 and 6.58 ?g/ml, respectively. The results showed that the affinities of RQ183 to the substrate and the primer were decreased dramatically. It is suggested that Arg182 and Arg183 were involved in the active site function of DNA polymerase ?, in binding to DNA template, in recognizing of primer, and in binding to and catalyzing of substrates of the enzyme.