Determination of half-reaction equilibrium in a ping-pong enzyme mechanism

Substituted enzyme (or ping-pong) mechanisms usually involve enzymes that exist in two forms that alternate during the catalytic reaction. A method is described here for determining the position of the equilibrium of a half reaction in a ping-pong enzyme mechanism that is based on the kinetics of the burst reaction which occurs upon addition of reactants that recycle the enzyme from one form to another. The theoretical basis for the analysis is developed, and the method is applied to the half reaction of the aldimine form of aspartate transaminase with difluoro-oxaloacetate. Special issue dedicated to Herman Bachelard     

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.     

Properties of a purified nuclear topoisomerase from L1210 cells

A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.     

Induction of apoptosis and c-myc in L1210 lymphocytic leukemia cells by adenosine

High concentrations of adenosine (Ado), when added to L1210 lymphocytic leukemia cells, resulted in apoptosis or programmed cell death. The apoptotic process was accompanied by distinct morphological changes including chromatin condensation and blebbing of plasma membranes. Extensive DNA fragmentation was correlated with Ado concentrations. Furthermore, apoptosis in these cells was preceded by an early but transient expression of c-myc proto-oncogene, and was not influenced by homocysteine thiolactone added to the cells. Since severe combined immunodeficiency (SCID) is associated with a deficiency of adenosine deaminase, leading to defects in both cellular and humoral immunity, Ado-induced apoptosis may thus be a contributing factor in the pathology of SCID.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Kinetic mechanism of NADP-malic enzyme from maize leaves

The kinetic mechanism of NADP-dependent malic enzyme purified from maize leaves was studied in the physiological direction. Product inhibition and substrate analogues studies with 3 aminopyridine dinucleotide phosphate and tartrate indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism. NADP is the leading substrate followed by l-malate and the products are released in the order of CO₂, pyruvate and NADPH. The enzyme also catalyzes a slow, magnesium-dependent decarboxylation of oxaloacetate and reduction of pyruvate and oxaloacetate in the presence of NADPH to produce l-lactate and l-malate, respectively.     

Kinetic mechanism of choline kinase from rat striata

The kinetic mechanism of choline kinase associated with both the cytosolic and membrane fractions of synaptosomes isolated from rat striata was studied. The velocity of choline kinase was measured using various concentrations of MgATP at several concentrations of uncomplexed Mg2+ and a single concentration of choline. This experiment was repeated using different concentrations of choline. Analysis of these data according to a terreactant mechanism indicates that MgATP binds in rapid equilibrium prior to Mg2+, but the binding of MgATP and choline is random. Product inhibition by phosphorylcholine was noncompetitive versus both choline and MgATP. Hemicholinium-3 (HC-3), an analog of choline and competitive inhibitor of the sodium-dependent high affinity choline transport system, was noncompetitive versus choline and uncompetitive versus MgATP at high levels of Mg2+. However, when the concentration of Mg2+ was decreased below the KMg2 +, HC-3 was noncompetitive versus MgATP. Thiocholine, another analog of choline, gave slope-linear intercept hyperbolic inhibition versus choline. Mg-5'-adenylyl imidodiphosphate, an analog of MgATP, was competitive versus MgATP and noncompetitive versus choline. Virtually identical results were obtained using either soluble or particulate forms of choline kinase from rat striata. All data are consistent with the mechanism suggested by initial velocity studies alone and additionally suggest that the release of MgADP is slow, occurs last, and may limit the overall rate of the reaction.     

Enantiomeric selectivity of adenosine transport systems in mouse erythrocytes and L1210 cells.

In mediating the entry of adenosine into mouse erythrocytes and mouse leukaemia L1210 cells, nucleoside transport systems were stereoselective, showing a marked preference for the D-enantiomer of adenosine (D-Ado). Inward zero-trans fluxes of the mirror-image isomer, L-adenosine (L-Ado), in those cells were slow relative to those of D-Ado. Contributing to L-Ado fluxes in both cell types were (i) a transporter-mediated process of high nitrobenzylthioinosine-sensitivity and (ii) simple diffusion.     

Ribonuclease-resistance patterns of RNA from L1210 cells.

Ribonuclease resistance of RNA from mammalian cells was studied as a possible measure of the extent of base pairing. Ribonuclease-sensitive and resistant components of the RNA were discerned from the biphasic kinetics of the ribonuclease reaction. The amount of resistant component increased progressively with increasing salt concentration until it comprised 20–40% of the total RNA. Comparisons between different RNA fractions from L1210 cells revealed differences in the content of resistant component, in the order: nucleolar ribosomal-precursor RNA > mature ribosomal RNA > nucleoplasmic RNA. In each RNA fraction, the amount of resistant component was greater when measured by a cytidine label than by a uridine label. Comparison with known double-stranded RNA indicated that the bulk of the resistant component consists of lessstable structures, such as short or imperfect helices.     

Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.     

5'-Nucleotidase of L 1210 cells. Cytochemical and kinetic studies.

The activity of 5'-nucleotidase in L 1210 leukaemia cells fixed in situ by glutaraldehyde was established by electron microscopic cytochemistry and biochemical assays. The enzyme activity is localized uniquely on the cell surface. In biochemical assays the time course of phosphate liberation by the suspension of glutaraldehyde fixed cells was linear up to 15 min incubation. The activity was proportional to the number of cells in the suspension, amounting of 0.019 units activity per 10(8) cells.     

Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells

Intact murine L1210 leukemic cells incorporated significant quantities of [³H]-N-acetylneuraminic acid directly from CMP-N-acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N-acetyl-neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP-N-acetyl-neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N-acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP-N-acetylneuraminic acid during incubations with L 1210 cells was rather insignificant. Electron microscope autoradiography of cells incubated with CMP-N-acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP-N-acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells. Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP-[³H]-galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP-[³H]-galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L-1210 cells incubated in UDP-[³H]-galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto-syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.     

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.