Kinetic and expression profiling of cytosolic pyruvate kinase enzyme during seed development of Indian mustard (Brassica juncea)

In the recent years tremendous progress has been achieved in deconstructing the oil biosynthetic pathways, majority of which is in Arabidopsis. Glycolysis is fundamental to this process as it is the cardinal supplier of precursors for fatty acid metabolism. Recent reports suggest that modification and expression of pyruvate kinase (PK), a crucial regulatory enzyme involved in glycolysis is one of the plausible ways to alter seed oil content in plants. In the present study we evaluated the kinetic behavior and expression profiling of pyruvate kinase, associated with seed development in a major oilseed crop B. juncea. Developmental profiling of the enzyme showed that enzyme activity was highest during middle stage (35 DAF) of seed development which is strongly corroborated by the expression profiling of the enzyme using RT-PCR approach. Oil accumulation pattern also correlated with the enzyme expression study. Comparative activity profiling from different tissues showed seedlings to have elevated activity than other tissues. For kinetic characterization, the enzyme was partially purified by 12.3 fold using DEAE-Sephadex column and showed a narrow pH optimum of 7.0. In presence of saturated substrate concentration, the enzyme exhibited hyperbolic kinetics for both ADP and PEP with Km (Michaelis constant value) for PEP and ADP was found to be 178.5 and 96.45 μM respectively. ATP and citrate are the most significant allosteric effectors of the partially purified PK. Study on isozymes of PK resulted in a single band.     

Kinetic model of laccase-catalyzed oxidation of aqueous phenol

Laccase from Trametes versicolor (EC 1.10.3.2) catalyzes the oxidation of aqueous phenol by oxygen and has demonstrated good potential for applications in various industrial and environmental processes. A kinetic model of this system has been developed to facilitate a better understanding of the mechanisms and rate-limiting steps of enzyme-catalyzed transformation and to eventually assist in the choice and design of suitable reactor systems. A kinetic model was derived based on the differential and mass balance equations that describe the interactions of various forms of the enzyme with the aromatic substrate and oxygen. This model also incorporated an expression accounting for enzyme inactivation over time due to the reaction environment. The model was validated by comparing model predictions with experimental observations of phenol transformation and oxygen consumption over time at a variety of enzyme concentrations. Excellent agreement was found between experimental data and predictions of the kinetic model. Sensitivity analyses demonstrated that the reaction between oxidized-laccase and phenol was the rate-limiting step.     

Expression of rat hepatic glucokinase in Escherichia coli

Rat liver glucokinase was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The expressed protein starts with the predicted initiator methionine residue and ends at the appropriate carboxyl terminal residue. It was partially purified by ammonium sulfate precipitation and gel filtration and had kinetic and physical properties similar to the purified rat liver enzyme. The efficient expression of this low abundance hepatic protein in bacteria provides a system for in vitro analysis of mutations of the enzyme.     

Kinetic modeling of hyperpolarized 13C label exchange between pyruvate and lactate in tumor cells

Measurements of the kinetics of hyperpolarized (13)C label exchange between [1-(13)C]pyruvate and lactate in suspensions of intact and lysed murine lymphoma cells, and in cells in which lactate dehydrogenase expression had been modulated by inhibition of the PI3K pathway, were used to determine quantitatively the role of enzyme activity and membrane transport in controlling isotope flux. Both steps were shown to share in the control of isotope flux in these cells. The kinetics of label exchange were well described by a kinetic model that employed rate constants for the lactate dehydrogenase reaction that had been determined previously from steady state kinetic studies. The enzyme showed pyruvate inhibition in steady state kinetic measurements, which the kinetic model predicted should also be observed in the isotope exchange measurements. However, no such pyruvate inhibition was observed in either intact cells or cell lysates and this could be explained by the much higher enzyme concentrations present in the isotope exchange experiments. The kinetic analysis presented here shows how lactate dehydrogenase activity can be determined from the isotope exchange measurements. The kinetic model should be useful for modeling the exchange reaction in vivo, particularly as this technique progresses to the clinic.     

Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5' to an abasic site in double-stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.     

粘质沙雷氏菌H3010发酵型D-乳酸脱氢酶基因的克隆表达、纯化及酶学性质研究

通过PCR技术从粘质沙雷氏菌H3010基因组DNA中扩增出该D-乳酸脱氢酶基因,连接至pET-28a（＋）表达载体,转入大肠杆菌BL21（DE3）中进行了重组表达,优化了酶纯化的条件,并对其酶学性质进行初步研究。结果表明,获得的该酶编码基因全长993bp,编码330个氨基酸,大小为37kDa。经优化表达及纯化条件后重组酶纯度可达90％。酶学性质研究发现,该重组酶最适反应温度为60℃,最适酶促反应pH为7．5（O．2mol／L磷酸盐缓冲液）,37℃下测得对底物丙酮酸的动力学参数Km=3．39mmol／L,Vmax=6．87mmol／（mg·min）,对辅酶NADH的动力学参数Km=1．43mmol／L,Vmax=1．61mmo]／（mg·min）。为酶法生产D-乳酸及利用代谢工程构建产D-乳酸的基因工程菌打下基础。     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

Polyphosphate kinase from M. tuberculosis: an interconnect between the genetic and biochemical role

The enzyme Polyphosphate Kinase (PPK) catalyses the reversible transfer of the terminal γ-Pi of ATP to form a long chain Polyphosphate (PolyP). Using an IPTG inducible mycobacterial vector, the vulnerability of this gene has been evaluated by antisense knockdown experiments in M. tuberculosis. Expression profiling studies point to the fact that down regulation of PPK caused cidality during the late phase in contrast to its bacteriostatic mode immediately following antisense expression. PPK thus seems to be a suitable anti-tubercular drug target. The enzyme which is a tetramer has been cloned in E. coli and purified to homogeneity. An enzyme assay suitable for High Throughput Screening was optimized by using the statistical Taguchi protocol and the kinetic parameters determined. The enzyme displayed a strong product inhibition by ADP. In order to accurately estimate the product inhibition, progress curve analysis of the enzyme reaction was monitored. The kinetic equation describing the progress curve was suitably modified by taking into account the product inhibition. The reversible nature of the enzyme indicated a possibility of a two way ATP↔ADP switch operating in the bacteria as a response to its growth requirement.     

Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA) to the free amino group of arylamines and hydrazines. Previous studies have reported that overexpression of NAT from Mycobacterium smegmatis and Mycobacterium tuberculosis may be responsible for increased resistance to the front-line antitubercular drug, isoniazid, by acetylating and hence inactivating the prodrug. We report the kinetic characterization of M. tuberculosis NAT which reveals that substituted anilines are excellent substrates but that isoniazid is a very poor substrate for this enzyme. We propose that the expression of NAT from M. tuberculosis (TBNAT) is unlikely to be a significant cause of isoniazid resistance. The kinetic parameters for a variety of TBNAT substrates were examined, including 3-amino-4-hydroxybenzoic acid and AcCoA, revealing K m values of 0.32 +/- 0.03 and 0.14 +/- 0.02 mM, respectively. Steady-state kinetic analysis of TBNAT reveals that the enzyme catalyzes the reaction via a bi-bi ping-pong kinetic mechanism. The pH dependence of the kinetic parameters reveals that one enzyme group must be deprotonated for optimal catalytic activity and that two amino acid residues at the active site of the free enzyme are involved in binding and/or catalysis. Solvent kinetic isotope effects suggest that proton transfer steps are not rate-limiting in the overall reaction for substituted aniline substrates but become rate-limiting when poor hydrazide substrates are used.     

Soluble expression and purification of porcine pepsinogen from Pichia pastoris

This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).     

The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.     

Overexpression, purification and characterization of the Trichoderma atroviride endochitinase, Ech42, in Pichia pastoris

The endochitinase gene ech42 from Trichoderma atroviride was cloned and expressed in Pichia pastoris using a constitutive expression system. Over 98% of the recombinant protein was secreted into the culture medium as glycoprotein. A high endochitinase concentration, 186 mg/L with a specific enzyme activity of 14,128 Umg(-1) was produced. The optimal enzyme kinetic parameters for the recombinant protein were identical to those reported for the enzyme isolated from T. atroviride. The recombinant endochitinase possesses suitable features for biotechnological applications, such as activity at acidic pH and thermostability.     

Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.

Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.     

Human caspases in vitro: expression, purification and kinetic characterization

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.     

Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli

The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector. The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants. The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl-modifying reagents. The results demonstrate that a protein with an Mr of 70,000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity. Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.     

Hysteresis and positive cooperativity of iceberg lettuce polyphenol oxidase.

A kinetic study of the diphenolase activity of latent polyphenol oxidase (PPO), purified from Iceberg lettuce (Lactuca sativa L), revealed a sigmoid relationship between the reaction rate and the substrate concentration with a high Hill coefficient (n(H) = 3.8). This positive cooperativity had not been previously described for any PPO. Furthermore, the enzyme showed a lag phase in the expression of this activity, suggesting a hysteretic nature of the enzyme. The kinetic behavior, the latency and the lag phase varied at different steps of the purification process. PPO showed hyperbolic or cooperative kinetics depending on the pH assay and the sodium dodecyl sulfate (SDS) concentration. Substrate-induced slow conformational change of the oligomeric enzyme is suggested. The conformational change would be toward a more active enzyme form with higher affinity for the substrate and favoured by acid pH and SDS.     

Genetic mechanism of cis-AB inheritance. I. A case associated with unequal chromosomal crossing over.

In contradiction to the general Mendelian inheritance of blood group ABO expression, the A and B characteristics are inherited together from one parent in the rare Cis-AB phenotype. Since the synthesis of blood group A and B substances are controlled by N-acetylgalactosoaminyltransferase (A-enzyme) and galactosyltransferase (B-enzyme), the genetic mechanism of Cis-AB expression may be elucidated by examining the characteristics of A- and B-enzymes in Cis-AB plasma. Biochemical study reveals that the examined Cis-AB plasma contains two separable enzyme components: one with kinetic properties similar to those of common A2-enzyme, but differing from A1-enzyme, and another with kinetic characteristics similar to those of common B-enzyme. Therefore, Cis-AB expression, at least in the case examined, is due to unequal crossing over, producing a chromosome with alleles for A2- and B-enzymes, rather than to a structural mutation in A or B alleles producing a single abnormal enzyme with bifunctional activity.     

Kinetic mechanism for stimulation by monovalent cations of the amidase activity of the plasma protease bovine activated protein C

A study of the effect of monovalent cations on the steady-state kinetic parameters for the hydrolysis of the synthetic substrate N alpha-benzoyl-L-arginine-p-nitroanilide by activated bovine plasma protein C (APC) has been undertaken. The enzyme displayed a strict requirement for monovalent cations in its expression of amidolytic activity toward this substrate. Analysis of the variation in initial hydrolytic reaction rates, as a function of metal ion concentrations, suggested that at least two cation sites, or classes of sites, were necessary for catalysis to occur. After examination of the rate equations consequential to many different enzymic mechanisms that could account for these kinetic data, a mechanism was developed that fit the great majority of the experimental observations. In this mechanism it is postulated that cations bind to the enzyme in pairs, with a kinetically observable single binding constant, either preceded by or followed by binding of substrate. Catalysis occurs only after the enzyme-(metal cation)2-substrate complex is assembled. Some physical support for this mechanism was obtained upon the discovery that the binding (dissociation) constant for a competitive inhibitor of APC, p-aminobenzamidine, as determined by kinetic methodology, was independent of the concentration of Na+ and Cs+.     

Expression and Refolding of Tobacco Anionic Peroxidase from E. coli Inclusion Bodies

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.