Initial Velocity Studies Research Articles

Kinetic and catalytic mechanism of the methionine-derived glucosinolate biosynthesis enzyme methylthioalkylmalate synthase

In Brassica plants, methionine-derived aliphatic glucosinolates are chemically diverse natural products that serve as plant defense compounds, as well as molecules with dietary health-promoting effects. During their biosynthesis, methylthioalkylmalate synthase (MAMS) catalyzes the elongation reaction of the aliphatic chain. The MAMS catalyzed condensation of 4-methylthio-2-oxobutanoic acid (4-MTOB) and acetyl-CoA generates a 2-malate derivative that either enters the pathway for synthesis of C3-glucosinolates or undergoes additional extension reactions, which lead to C4- to C9-glucosinolates. Recent determination of the x-ray crystal structure of MAMS from Brassica juncea (Indian mustard) provided insight on the molecular evolution of MAMS, especially substrate specificity changes, from the leucine biosynthesis enzyme α-isopropylmalate synthase (IPMS) but left details of the reaction mechanism unanswered. Here we use the B. juncea MAMS2A (BjMAMS2A) isoform to analyze the kinetic and catalytic mechanism of this enzyme. Initial velocity studies indicate that MAMS follows an ordered bi bi kinetic mechanism, which based on the x-ray crystal structure, involves binding of 4-MTOB followed by acetyl-CoA. Examination of the pH-dependence of kcat and kcat/Km are consistent with acid/base catalysis. Site-directed mutagenesis of three residues originally proposed to function in the reaction mechanism - Arg89 (R89A, R89K, R89Q), Glu227 (E227A, E227D, E227Q), and His388 (H388A, H388N, H388Q, H388D, and H388E) - showed that only two mutants (E227Q and H388N) retained activity. Based on available structural and biochemical data, a revised reaction mechanism for MAMS-catalyzed elongation of methionine-derived aliphatic glucosinolates is proposed, which is likely also conserved in IPMS from leucine biosynthesis in plants and microbes.

Structural and Functional Characterization of Mycobacterium tuberculosis Homoserine Transacetylase.

Mycobacterium tuberculosis (Mtb) lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential S-adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated Mtb HTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization of Mtb HTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substrates─acetyl-CoA and l-homoserine─engage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA and O-acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractive Mtb target.

Steady-state kinetic analysis of halogenase-supporting flavin reductases BorF and AbeF reveals different kinetic mechanisms

The short-chain flavin reductases BorF and AbeF reduce FAD to FADH2, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed. AbeF and BorF can reduce FAD, FMN, and riboflavin in vitro and are selective for NADH over NADPH. Initial velocity studies in the presence and absence of inhibitors showed that BorF proceeds by a sequential ordered kinetic mechanism in which FAD binds first, while AbeF follows a random-ordered sequence of substrate binding. Fluorescence quenching experiments verified that NADH does not bind BorF in the absence of FAD, and that both AbeF and BorF bind FAD with higher affinity than FADH2. pH-rate profiles of BorF and AbeF were bell-shaped with maximum kcat at pH 7.5, and site-directed mutagenesis of BorF implicated His160 and Arg38 as contributing to the catalytic activity and the pH dependence.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi–bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi–bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

Catalytic studies of glutathione transferase from Clarias gariepinus (Burchell) in dilute and crowded solutions

Kinetic properties of purified Clarias gariepinus glutathione transferase (CgGST) was studied in the presence of Ficoll 70, Polyethylene glycol (PEG) 6000, bovine serum albumin (BSA) and in dilute solution. This was done to mimic the cytosol thereby unraveling the actual mechanism of detoxication involving glutathione transferase (GST) in the crowded intracellular milieu. CgGST from the liver of Clarias gariepinus was purified to homogeneity by affinity chromatography on glutathione (GSH) - agarose. Initial-velocity study was performed by varying the concentrations of GSH at various fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) and vice-versa. Data obtained were fitted to the three equations representing random-ordered, compulsory-ordered and ping-pong mechanisms to obtain kinetic parameters. Product inhibition studies using sodium chloride (NaCl) was done by varying the concentrations of NaCl and CDNB at a fixed concentration of GSH and vice-versa. Data obtained were fitted to three equations representing competitive, non-competitive and uncompetitive inhibitions to obtain the inhibition constants (KiGSH and KiCDNB). Optimal temperature of CgGST activity was 20 °C both in dilute and crowded solutions. Maximum velocity (Vmax) in dilute solution was decreased, while KmGSH and KmCDNB were increased in the presence of the crowding agents. Turnover number (kcat), catalytic efficiency - kcat/KmGSH,kcat/KmCDNB and inhibition constants – (KiGSH and KiCDNB) were reduced in crowded solutions. Mechanism of catalysis was steady – state random sequential in both dilute and crowded solutions. The study concluded that although the catalytic efficiency of the enzyme was reduced in crowded solution, mechanism of catalysis remains the same in both crowded and dilute solutions.

Kinetics mechanism and regulation of native human hepatic thymidine phosphorylase.

Thymidine phosphorylase (TP; EC 2.4.2.4) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective nucleobases and 2-deoxy-α-d-ribose-1-phosphate (dRib-1-P). TP is a key enzyme in the pyrimidine salvage pathways. Activity of the enzyme is crucial in angiogenesis, cancer chemotherapy, radiotherapy, and tumor imaging, Nevertheless, a complete set of kinetic parameters has never been reported for any human TP. This study describes the kinetic mechanism and regulation of native human hepatic TP. The liver is a main site of pyrimidine metabolism and contains high levels of TP. Initial velocity and product inhibition studies demonstrated that the basic mechanism of this enzyme is a sequential random bi-bi mechanism. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation, and a binding pattern indicating that the binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KThymidine = 284 ± 55, KPi = 5.8 ± 1.9, KThymine = 244 ± 69, and KdRib-1-P = 90 ± 33 μM. Thymine was a product activator, but becomes a substrate inhibitor at concentrations eight times higher than its Km. dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bounded better to the Enzyme●Pi complex than the free enzyme, but had better affinity to the free enzyme than the Enzyme●Thymidine complex. In the reverse reaction, dRib-1-P enhanced the binding of thymine. The enhancement of the thymine binding along with the fact that dRib-1-P was a non-competitive product inhibitor suggests the presence of another binding site for dRib-1-P on the enzyme.

Kinetic Mechanism of Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide, pyridine, and other structural analogues. Aberrantly increased NNMT activity results in the depletion of SAM, nicotinamide (NAM), and nicotinamide adenine dinucleotide (NAD+); NAM is required for NAD+ biosynthesis. SAM depletion impairs the methylation potential of the cell, resulting in hypomethylated histones and an altered epigenetic profile. In addition, decreased NAD+ levels negatively affect energy metabolism by disrupting oxidative phosphorylation. Because of its impact on epigenetic states and NAD+ levels, NNMT is implicated in cancer, neurodegenerative diseases, and metabolic diseases, making it an appealing target for therapeutic intervention. To gain insights that would guide the design of inhibitors and activity-based probes, we performed detailed kinetic studies of human NNMT. Herein, we report the kinetic mechanism of NNMT. Our initial velocity, product inhibition, and dead-end analogue inhibition studies collectively indicate that NNMT uses a rapid equilibrium ordered mechanism, where NNMT first binds SAM, which is followed by NAM. Methyl transfer occurs, and methylated NAM and S-adenosylhomocysteine are released consecutively.

Purification characterization and inhibition studies on Phospholipase C from <i>Opisthacanthus capensis</i> (Black Scorpion) venom

Phospholipase C from Opisthacanthus capensis venom was partially purified and characterized. The enzyme was purified 105.2 fold with an overall yield of 13% using various steps involving ammonium sulphate precipitation, protamine sulphate treatment, sephadex G-75 fractionation and DE-52 anion exchange chromatography. The purified enzyme was homogeneous by the criteria of SDS polyacrylamide gel electrophoresis with a molecular weight of 29 kDa. The phospholipase C has pH and temperature optima of 7.2 and 600C, respectively with activation energy of 25KJ/mol and t1/2 of 1.50 hr. Initial velocity studies on Opisthacanthus capensis venom phospholipase C revealed a KM of 0.02 mM and Vmax of 0.015 µmol/min respectively. Studies on the effect of pH on KM and Vmax gave PKa1 of 6.9 and PKa2 of 7.4 with enthalpy of ionization of 20 KJ/mol suggesting histidine in the active site. The enzyme was positively modulated by Mg2+, Zn2+ and Ca2+ and negatively by Fe2+. While Hg2+ produced complete inhibition. Various concentrations of leaf aqueous extract of Momordica charantia also inhibited the activity of Opisthacanthus capensis venom phospholipase C in vitro with a display of competitive

An evaluation of glutathione transferaseassociated with Dichlorvos degradation inAfrican palm weevil (Rynchophorus phoenicis) larva

AbstractThis study was conducted to investigate the metabolic defensive mechanism in the larvae of African palm weevil (Rynchophorus phoenicis) administered with dichlorvos (2,2-dichlorovinyl dimethylphosphate) solution. Bioassay experiment with dichlorvos was conducted on the larva and glutathione-utilizing enzyme activities were determined in the major organs: fat body, gut, and head of R. phoenicis larva 48 h after treatment with 0–0.060 μg g−1 body weight dichlorvos solution. Glutathione transferase was purified from the gut of larvae by ion-exchange chromatography on diethylaminoethyl-Sephadex A50 and affinity chromatography on glutathione-Sepharose 4B columns. The purified enzyme was homogenous as revealed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Initial velocity studies were carried out on the purified enzyme using standard procedures. Bioassay experiment indicated alterations of glutathione peroxidase, glutathione reductase, and glutathione transferase activities in the major o...

Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans

The Pden_2689 gene encoding FerA, an NADH:flavin oxidoreductase required for growth of Paracoccus denitrificans under iron limitation, was cloned and overexpressed as a C-terminally His6-tagged derivative. The binding of substrates and products was detected and quantified by isothermal titration calorimetry and fluorometric titration. FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, which was confirmed by a negative shift in the redox potential of FMN after addition of FerA. Initial velocity and substrate analogs inhibition studies showed that FerA follows a random-ordered sequence of substrate (NADH and FMN) binding. The primary kinetic isotope effects from stereospecifically deuterated nicotinamide nucleotides demonstrated that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. The crystal structure of FerA revealed a twisted seven-stranded antiparallel β-barrel similar to that of other short chain flavin reductases. Only minor structural changes around Arg106 took place upon FMN binding. The solution structure FerA derived from small angle X-ray scattering (SAXS) matched the dimer assembly predicted from the crystal structure. Site-directed mutagenesis pinpointed a role of Arg106 and His146 in binding of flavin and NADH, respectively. Pull down experiments performed with cytoplasmic extracts resulted in a negative outcome indicating that FerA might physiologically act without association with other proteins. Rapid kinetics experiments provided evidence for a stabilizing effect of another P. denitrificans protein, the NAD(P)H:acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin.

Catalysis of Silver catfish Major Hepatic Glutathione Transferase proceeds via rapid equilibrium sequential random Mechanism

Fish hepatic glutathione transferases are connected with the elimination of intracellular pollutants and detoxification of organic micro-pollutants in their aquatic ecosystem. The two-substrate steady state kinetic mechanism of Silver catfish (Synodontis eupterus) major hepatic glutathione transferases purified to apparent homogeneity was explored. The enzyme was dimeric enzyme with a monomeric size of 25.6 kDa. Initial-velocity studies and Product inhibition patterns by methyl glutathione and chloride with respect to GSH-CDNB; GSH-ρ-nitrophenylacetate; and GSH-Ethacrynic acid all conforms to a rapid equilibrium sequential random Bi Bi kinetic mechanism rather than steady state sequential random Bi Bi kinetic. α was 2.96 ± 0.35 for the model. The pH profile of Vmax/KM (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pKa value of 6.88 and 9.86. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer and near neutral GST. The enzyme poorly conjugates 4-hydroxylnonenal and cumene hydroperoxide and may not be involved in oxidative stress protection. The seGST is unique and overwhelmingly shows characteristics similar to those of homodimeric class Pi GSTs, as was indicated by its kinetic mechanism, substrate specificity and inhibition studies. The rate- limiting step, probably the product release, of the reaction is viscosity-dependent and is consequential if macro-viscosogen or micro-viscosogen.

Abstract 2144: Kinetic mechanism of the lysine methyltransferase SMYD3 using MAP3K2 protein substrate

Abstract Dysregulation of DNA and histone methylation has been shown to play fundamental roles in carcinogenesis. Recently however, protein methylation in the cytoplasm has been linked to oncogenic Ras signaling. Mazur and colleagues demonstrated that SMYD3 (SET and MYND domain containing protein 3), a primarily cytoplasmic protein methyltransferase, can methylate MAP3K2 using S-adenosyl-L-methionine (SAM) as a cofactor (Mazur et al. 2014. Nature 510(7504):283-7). Methylation by SMYD3 preserves the active, phosphorylated state of MAP3K2 sustaining activation of the Ras/Raf/MEK/ERK/ signaling module and potentiating formation of Ras-driven cancers. Here, we characterized the kinetic mechanism of SMYD3 using MAP3K2 protein as a substrate. SMYD3 activity was measured with a radiometric assay using 3H-SAM. Initial velocity, product and dead-end inhibitor studies indicate that SMYD3 uses a random kinetic mechanism where both SAM and MAP3K2 can bind apo-SMYD3. This was supported by substrate and ligand binding assays using SPR. Together, these studies elucidate the kinetic mechanism of SMYD3 with MAP3K2 and provide a context for inhibitor development. Citation Format: Thomas V. Riera, Tim J. Wigle, Jodi Gureasko, P. Ann Boriack-Sjodin, Robert A. Copeland. Kinetic mechanism of the lysine methyltransferase SMYD3 using MAP3K2 protein substrate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2144. doi:10.1158/1538-7445.AM2015-2144

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine.

Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo=0.002±0.0002 mM, KATP=0.05±0.008 mM, and Vmax=920±35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.

Kinetic Mechanism of Protein N-terminal Methyltransferase 1

The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors.

Characterization of the recombinant succinic semi-aldehyde dehydrogenase from Saccharomyces cerevisiae.

The yeast succinic semi-aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS-PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µM NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi-aldehyde (Km = 15.48 ± 0.14 µM) and could use both NAD(+) and NADP(+) as co-factors, with Km values of 579.06 ± 30.1 µM and 1.017 ± 0.46 mM, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD(+) (Ki = 129.5 µM) but a non-competitive inhibitor with respect to succinate semi-aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with Ki = 1.13-10.2 mM, and showed competitive inhibition with respect to NAD(+) and mixed-competitive, non-competitive and non-competitive inhibition, respectively, with respect to succinate semi-aldehyde. The kinetic data suggest a 'ping-pong' mechanism.

Magnetic clouds and origins in STEREO era

AbstractWhen a coronal mass ejection (CME) encounters the Earth, the Earth's electromagnetic environment is disturbed, especially when it is a magnetic cloud (MC) with enhanced, steady, and long‐lasting southward field. The speed and the magnetic field of an MC are the two important properties for its geoeffectiveness. The correspondence between a CME and its resulting MC is not straightforward, partly due to the CME velocity and the complications during propagation through corona and the solar wind. From 2007 to 2012, we have three observing points at 1 AU near the ecliptic plane (ACE and STEREO A and B). We search for MC events encountered at one of the three observers and study the statistics independently and in comparison. We found that the annual number of MCs at each receiver varies significantly and the temporal variation at each receiver does not always follow the solar activity level. The speed and the magnetic field strength of the MCs do vary with the solar activity level. The polarity of MC magnetic field at ACE and STEREO also shows large fluctuations. We have also identified the CME and solar activity sources for the L1 MC events. STEREO SECCHI images served critical roles in the determination of the CMEs both in solar quiet times and active times. We found that halo CMEs are not necessarily good indicators for receiving MCs. Further studies of CME initial velocity and the propagation through the heliosphere are needed in order to improve our space weather forecasting capability.

Kinetic and structural analysis of two transferase domains in Pasteurella multocida hyaluronan synthase

Abstract Pasteurella multocida hyaluronan synthase (PmHAS) encompasses two transferase domains that elongate a growing hyaluronan (HA) oligosaccharide chain by addition of either GlcNAc or GlcUA residues from a corresponding UDP-sugar. Initial velocity studies of single-step elongations were conducted for both domains by independently varying the concentrations of the HA oligosaccharide and the UDP-sugar. Two-substrate models were discriminated by their goodness-of-fit parameters and by dead-end inhibition studies. A mechanistic shift from a steady-state ordered bi-bi to rapid equilibrium ordered bi-bi mechanism was observed at the NAc-site between the HA6 and HA8 elongation. This shift was invoked by a minor reduction in turnover number kcat. Both NAc- and UA-transferase domains follow a sequential kinetic mechanism, most likely an ordered one in which the UDP-sugar donor binds first, followed by the HA oligosaccharide. After transfer of the sugar moiety, both products are released, first the elongated HA oligosaccharide and then the UDP sugar. This mechanism was visualized with a structural model of PmHAS that presented two flexible loops, one in each transferase domain; these loops form a bridge above the active site. Based on structural similarities between PmHAS with α1,3-galactosyltransferase (α3GT) and β1,4-galactosyltransferase (β4Gal-T1), these flexible loops are seemingly involved in a conformational change upon binding the UDP-sugar, inducing the ordered mechanism. Kinetic analyses have demonstrated significant differences in dissociation constant KD,UA and KM,NAc of 124 ± 31 μM and 28 ± 8.8 mM, respectively, suggesting that the variance in the binding affinity of the two UDP-sugars controls the molecular weight of hyaluronan.

Kinetic and pH studies on human phenylethanolamine N-methyltransferase

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-l-methionine (AdoMet) is converted to S-adenosyl-l-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the logV pH-profile was not well defined, plots of logV/K versus pH for AdoMet and phenylethanolamine, as well as the pKi versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.

Mechanism and Regulation of Mycobactin Fatty Acyl-AMP Ligase FadD33

Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.

Substrate Specificity, Processivity, and Kinetic Mechanism of Protein Arginine Methyltransferase 5

Protein arginine methyltransferases (PRMTs) have emerged as attractive therapeutic targets for heart disease and cancers. PRMT5 is a particularly interesting target because it is overexpressed in blood, breast, colon, and stomach cancers and promotes cell survival in the face of DNA damaging agents. As the only known member of the PRMT enzyme family to catalyze the formation of mono- and symmetrically dimethylated arginine residues, PRMT5 is also mechanistically unique. As a part of a program to characterize the mechanisms and regulation of the PRMTs and develop chemical probes targeting these enzymes, we characterized the substrate specificity, processivity, and kinetic mechanism of bacterially expressed Caenorhabditis elegans PRMT5 (cPRMT5). In this report, we demonstrate that distal positively charged residues contribute to substrate binding in a synergistic fashion. Additionally, we show that cPRMT5 catalyzes symmetric dimethylation in a distributive fashion. Finally, the results of initial velocity, product, and dead-end inhibition studies indicate that cPRMT5 uses a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes. In total, these studies will guide PRMT5 inhibitor development and lay the foundation for studying how the activity of this medically relevant enzyme is regulated.