Enzyme Stoichiometry Research Articles

Cheaters, diffusion and nutrients constrain decomposition by microbial enzymes in spatially structured environments

AbstractExtracellular enzymes allow microbes to acquire carbon and nutrients from complex molecules and catalyse the rate‐limiting step in nutrient mineralization. Because the factors regulating enzyme production are poorly understood, I used a simulation model to examine how competition, nutrient availability and spatial structure affect microbial growth and enzyme synthesis. In simulations where enzyme‐producing microbes competed with cheaters (who do not synthesize enzymes but take‐up product), higher enzyme costs favoured cheaters, while lower rates of enzyme diffusion favoured producers. Cheaters and producers coexisted in highly organized spatial patterns at intermediate enzyme costs and diffusion rates. Simulations with varying nutrient inputs showed that nitrogen supply can limit carbon mineralization, microbial growth and enzyme production because of the nitrogen‐demanding stoichiometry of enzymes (C : N = c. 3.5 : 1). These results suggest that competition from cheaters, slow diffusion and nitrogen limitation may constrain microbial foraging and the enzymatic decomposition of complex compounds in natural environments.

Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase β (pol β), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol β, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol β, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol β may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol β. This enables pol β to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.

The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.

Kinetics of thapsigargin-Ca(2+)-ATPase (sarcoplasmic reticulum) interaction reveals a two-step binding mechanism and picomolar inhibition.

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca(2+)-ATPase interaction in the absence and presence of non-ionic detergent. The observed "off" rate constant was measured as 0.0052 s-1 at 26 degrees C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca2+. The observed "on" rate constant of thapsigargin was measured at 26 degrees C as 25 s-1 irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca(2+)-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26 degrees C.

Direct Involvement of Intracellular Ca2+ Transport ATPase in the Development of Thapsigargin Resistance by Chinese Hamster Lung Fibroblasts

Thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCA), inhibits cell proliferation when added to culture media in the nanomolar concentration range. However, long term exposure to gradually increasing concentrations of TG induces resistance to TG inhibition in both the parental Chinese hamster lung fibroblast DC-3F and a subline derived from it via transfection and stable expression of a full-length cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance develops in parallel with selection of cells expressing higher levels of the endogenous SERCA2 as well as of the exogenous transfected SERCA1 ATPase, whose Ca2+ transport function can be studied in situ by imaging techniques and following isolation in microsomal fractions. Microsomes isolated from resistant cells contain two functionally distinct populations of ATPases: a population that is inhibited by stoichiometric titration with TG, and a population displaying resistance to inhibition even when TG exceeds the enzyme stoichiometry. It is apparent that resistance to TG develops in parallel with (a) selection of cells expressing high levels of SERCA ATPases, and (b) selection of an ATPase that is resistant to TG.

Rational design of quinazoline-based irreversible inhibitors of human erythrocyte purine nucleoside phosphorylase

Described herein is the rational design of irreversible inhibitors of human erythrocyte purine nucleoside phosphorylase (PNPase). Inhibitor design started with the observation that the amino group of 8-aminoquinazolin-4(3H)-one interacts with enzyme-bound phosphate. This observation correctly predicted that the 5,8-dione (quinone) and 5,8-dihydroxy (hydroquinone) derivatives of quinazolin-4(3H)-ones would enter the active site. The amine-phosphate interaction also served to confirm that a quinazolin-4(3H)-one binds in the PNPase active sites like a purine substrate. From models of the PNPase active site it was possible to design quinazoline-based quinones that undergo a reductive-addition reaction with an active-site glutamate residue. The best inhibitor studied, 2-(chloromethyl)quinazoline-4,5,8(3H)-trione, rapidly inactivates PNPase by a first-order process with an inhibitor to enzyme stoichiometry of 150. The active-site hydroquinone adduct of this inhibitor eliminates a leaving group to afford a quinone methide species positioned to alkylate another active-site glutamate residue. Thus, this inhibitor is designed to cross-link the PNPase active site by reductive addition followed by the generation of an alkylating quinone methide species.

Use of monoclonal antibodies in immuno-electron microscopy for the determination of subunit stoichiometry in oligomeric enzymes: There are three α-subunits in the F 1-ATPase of Escherichia coli

The subunit stoichiometry of oligomeric enzymes can be determined by immuno-electron microscopy using monoclonal antibodies against the individual subunits. Monoclonal antibodies against native F 1-ATPase of Escherichia coli were prepared that were specific for the α-subunit. The immune complexes of F 1 and monoclonal antibodies were isolated. Electron microscopy revealed the presence of three irnmunoglobulins per molecule of F 1-ATPase. This unequivocally demonstrates an α 3 stoichiometry for the F 1-ATPase of E. coli.

H+/ATP stoichiometry for the gastric (K+ + H+)-ATPase.

The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150 mM KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639 +/- 84 and 619 +/- 65 nmol/min x mg protein, respectively, giving an H+/ATP ratio of 1.03 +/- .07. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (less than 10 nmol/min x mg protein) was exhibited by vesicles in 159 mM NaCl at pH 6.1 despite ATP hydrolysis of 187 +/- 46 nmol/min x mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H+/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K+ greater than 10 mM. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H+/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K+ + H+)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.

Hydroxyl‐Ion‐Induced Subunit Dissociation of Yeast Cytoplasmic Pyruvate Decarboxylase

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.