Evidence for sequential action of two ATPase active sites in yeast Msh2-Msh6

Bacterial MutS homodimers contain two ATPase active sites that have non-equivalent functions in DNA mismatch repair. The homologous Msh2-Msh6 complex in eukaryotes also has intrinsic ATPase activity that is essential for mismatch repair. Here, we investigate differences in the two putative ATPase active sites by examining the properties of heterodimers containing alanine substituted for an invariant glutamic acid in the active site of either Msh2, Msh6 or both. Mutation rates in wild type versus Glu-->Ala mutant haploid yeast strains indicate that both ATPase active sites are essential for mismatch repair activity in vivo. The properties of purified heterodimers suggest that the ATPase active site in Msh6 binds ATP with higher affinity and hydrolyzes ATP faster and with higher efficiency than does the ATPase active site in Msh2. This suggests sequential action of the two ATPase active sites, in which ATP binds to Msh6 first to trigger downstream events in mismatch repair.     

Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.     

Restricted active site docking by enzyme-bound substrate enforces the ordered cleavage of prothrombin by prothrombinase

The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.     

Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers.

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.     

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.     

Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (alpha) and R2 (beta). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited alpha(4)beta(4) complex in the presence of dATP and an active alpha(2)beta(2) complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30-50 times stronger in the alpha(4)beta(4) complex than in the alpha(2)beta(2) complex, which was in equilibrium with free alpha(2) and beta(2) subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form alpha(4)beta(4) complexes. ATP could also induce the formation of a generally inhibited alpha(4)beta(4) complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for alpha(4)beta(4) formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive alpha(6)beta(2) complexes.     

Regulation and state of aggregation of Bacillus subtilis prephenate dehydratase in the presence of allosteric effectors.

Prephenate dehydratase from Bacillus subtilis was found to exist in three states of aggregation. A high molecular weight (210,000) species was fully active and the catalytic activity was unaffected by the effectors methionine or phenylalanine. Low concentrations of phenylalanine caused dissociation to a Mr = 55,000 dimer. Heating to 32 degrees C also caused dissociation, but cooling and adding substrate or methionine favored association. When no effectors were present the enzyme eluted from Sephadex columns as a monomer. Both methionine and phenylalanine shifted the equilibrium from the inactive monomer to the active dimeric enzyme. In the presence of a saturating methionine concentration, the dimer possessed the same high activity as did the 210,000-dalton form. Phenylalanine inhibited the dimer, but not the higher molecular weight form. A model involving only three types of sites (catalytic, association-activation, and inhibition) is consistent with the data. It is proposed that phenylalanine is the preferred metabolite for binding both effector sites on the dimer; it binds the association-activation site with higher affinity than the inhibition site, but binding at the latter site has a greater effect on the catalytic rate. Methionine, like phenylalanine, has a hydrophobic side chain but is accommodated only at the association-activation site.     

Biochemical and kinetic analysis of the RNase active sites of the integrase/tyrosine family site-specific DNA recombinases

In this study, we have used multiple strategies to characterize the mechanisms of the type I and type II RNA cleavage activities harbored by the Flp (pronounced here as "flip") site-specific DNA recombinase (Flp-RNase I and II, respectively). Reactions using half-sites pre-bound by step-arrest mutants of Flp agree with a "shared active site" being responsible for the type I reaction (as is the case with normal DNA recombination). In a "pre-cleaved" type I substrate containing a 3'-phosphotyrosyl bond, the Flp-RNase I activity can be elicited by either wild type Flp or by Flp(Y343F). Kinetic analyses of the type I reaction are consistent with the above observations and support the notion that the DNA recombinase and type I RNase active sites are identical. The type II RNase activity is expressed by Flp(Y343F) in a half-site substrate and is unaffected by the catalytic constitution of a Flp monomer present on a partner half-site. Reaction conditions that proscribe the assembly of a DNA bound Flp dimer have no effect on Flp-RNase II. These biochemical results, together with kinetic data, are consistent with the reaction being performed from a "non-shared active site" contained within a single Flp monomer. The Flp-related recombinase Cre, which utilizes a non-shared recombination active site, exhibits the type I RNA cleavage reaction. So far, we have failed to detect the type II RNase activity in Cre. Despite their differences in active site assembly, Cre functionally mimics Flp in being able to provide two functional active sites from a trimer of Cre bound to a three-armed (Y-shaped) substrate.     

Reaction sequences for (Na+ + K+)-dependent ATPase hydrolytic activities: new quantitative kinetic models

To delineate better the reaction sequence of the (Na+ + K+)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na+ + K+)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after Pi release. However, contrary to requirements of usual formulations, Pi is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after Pi release, but also requiring a minor alternative pathway with ATP adding after K+ binds but before Pi release. The fit to the data also reveals that Pi binds nearly as rapidly to E2 X K X ATP as to E2 X K, whereas ATP binds quite slowly to E2 X P X K: the site resembles a cul-de-sac with distal ATP and proximal Pi sites. For the K+-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and Pi (as inhibitor) decrease with higher KCl concentrations, and both Pi and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K+, and with exclusive binding of substrate vs. either Pi or TNP-ATP. The greater sensitivity of the phosphatase reaction to both Pi and arsenate is attributable to the weaker binding by the occluded-K+ enzyme form occurring in the (Na+ + K+)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E1/E2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E2 conformation as the active site for the phosphatase reaction.     

Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites,and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.     

Biphasic steady-state kinetics of myosin adenosine triphosphatase. Evidence for a substrate effector site

The steady-state kinetics of the K+, Ca2+, and Mg2+-activated adenosine triphosphatase (ATPase) activities of rabbit skeletal myosin were investigated in the substrate concentration range from 0.05 microM to 5 mM and found not to follow Michaelis-Menten kinetics but rather to display biphasic behavior. The Ca2+-ATPase activity of myosin chymotryptic subfragment-1 (S-1), which has only one active site, also exhibits biphasic kinetics, thus excluding the possibility that the biphasic behavior is caused by negative cooperativity between the two active sites of myosin. Myosin K+ and Mg2+-ATPase are both activated by 5'-adenyl methylenediphosphonate (AdoPP[CH2]P) in a competitive manner at high substrate concentrations; i.e. the maximal velocity observed at high substrate concentrations is independent of the AdoPP[CH2]P concentration. This result provides evidence for substrate activation via binding to a regulatory site. Pyrophosphate inhibits myosin ATPase in a competitive manner at low substrate concentrations and in an uncompetitive manner at high substrate concentrations, with the uncompetitive Ki being smaller than the competitive Ki; i.e. pyrophosphate binds more tightly to the effector site than to the active site.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

5-Chlorolevulinate modification of porphobilinogen synthase identifies a potential role for the catalytic zinc.

Porphobilinogen synthase (PBGS) is a Zn(II) metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA). The nitrogen of the first substrate ends up in the pyrrole ring of product (P-side ALA); by contrast, the nitrogen of the second substrate molecule remains an amino group (A-side ALA). A reactive mimic of the substrate molecules, 5-chlorolevulinate (5-CLA), has been prepared and used as an active site directed irreversible inhibitor of PBGS. Native octameric PBGS binds eight substrate molecules and eight Zn(II) ions, with two types of sites for each ligand. As originally demonstrated by Seehra and Jordan [(1981) Eur. J. Biochem. 113, 435-446], 5-CLA inactivates the enzyme at the site where one of the two substrate molecules binds, and modification at four sites per octamer (one per active site) affords near-total inactivation. Here we report that 5-CLA-modified PBGS (5-CLA-PBGS) can bind up to four substrate molecules and four Zn(II) ions. Contrary to the conclusion of Seehra and Jordan, we find that the preferential site of 5-CLA inactivation is the A-side ALA binding site. On the basis of the dissociation constants, the metal ion binding sites lost upon 5-CLA modification are assigned to the four catalytic Zn(II) sites. 5-CLA-PBGS is shown to be modified at cysteine-223 on half of the subunits. We conclude that cysteine-223 is near the amino group of A-side ALA and propose that this cysteine is a ligand to the catalytic Zn(II). The vacant substrate binding site on 5-CLA-PBGS is that of P-side ALA. We have used 13C and 15N NMR to view [4-13C]ALA and [15N]ALA bound to 5-CLA-PBGS. The NMR results are nearly identical to those obtained previously for the enzyme-bound P-side Schiff base intermediate [Jaffe et al. (1990) Biochemistry 29, 8345-8350]. It appears that, in the absence of the catalytic Zn(II), 5-CLA-PBGS does not catalyze the condensation of the amino group of the P-side Schiff base intermediate with the C4 carbonyl derived from 5-CLA. On this basis we propose that Zn(II) plays an essential role in formation of the first bond between the two substrate molecules.     

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.     

Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 A resolution.

We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).     

Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.     

Study of the complex between porcine plasmin and alpha2-macroglobulin (author's transl)]

Porcine plasmin (EC 3.4.21.7) is obtained from plasminogen activated by human urokinase. This enzyme can bind, in an equimolecular ratio, to an alpha2-macroglobulin isolated from porcine serum. The number of active sites of plasmin has been determined through a burst titration of nitroaniline during the presteady-state hydrolysis of an amide substrate (N-alpha-carbobenzoxy-L-arginine-p-nitroanilide). The kinetic constants relative to a very sensitive ester substrate (N-alpha-carbobenzoxy-L-lysine nitrophenylester) hydrolysis have been measured. The binding of plasmin to alpha2-macroglobulin results in a complete inhibition of proteolytic activity, a reduction of active sites number and a decrease of esterolytic activity towards this substrate. In the complex, the residual activity (about 60%) is protected from protein inhibitors. Absorbance difference spectra show that 1 mol of alpha2-macroglobulin binds 1 mol of plasmin and 2 mol of trypsin. When plasmin is first bound to alpha2-macroglobulin, only 1 mol of trypsin can gain access tothe second site without removing the plasmin, showing that a steric hindrance is implicated in the inhibition performed by alpha2-macroglobulin binding.     

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.     

A comprehensive model for the allosteric regulation of mammalian ribonucleotide reductase. Functional consequences of ATP- and dATP-induced oligomerization of the large subunit.

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a new, comprehensive, and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding, and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine-specific site (a-site) results in formation of an inactive tetramer, and ATP binding to the newly described hexamerization site (h-site) drives formation of active R1(6)R2(6) hexamer. In contrast, an earlier phenomenological model [Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 67, 71-98] (the "RT" model) ignores aggregation state changes and mistakenly rationalizes ATP activation versus dATP inhibition as reflecting different functional consequences of ATP versus dATP binding to the a-site. Our results suggest that the R1(6)R2(6) heterohexamer is the major active form of the enzyme in mammalian cells, and that the ATP concentration is the primary modulator of enzyme activity, coupling the rate of DNA biosynthesis with the energetic state of the cell. Using the crystal structure of the Escherichia coliR1 hexamer as a model for the mR1 hexamer, a scheme is presented that rationalizes the slow isomerization of the tetramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R1(2)R2(2) and R1(6)R2(6) are inconsistent with a proposed model for R2(2) docking with R1(2) [Uhlin, U., and Eklund, H. (1994) Nature 370, 533-539], and an alternative is suggested.