Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase.

Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.     

Binding of terbium(III) to yeast enolase

Several independent criteria indicate 2 mol of terbium(III) bind to yeast enolase in the absence of substrate-fluorescence titrations of enzyme and metal, effects on thermal stability and published ultrafiltration and inhibition experiments. These measurements also suggest the terbium binding sites are the same as those normally occupied by “conformational” magnesium. Terbium binds much more strongly than magnesium, however, and measurements of the kinetics of the absorbance change in the terbium-enzyme on adding excess EDTA suggest the terbium-enzyme dissociation constant is about $\text{1}\text{500}$ that of the magnesium-enzyme. Measurements of enzyme activity as a function of substrate concentration show that terbium permits no enzymatic activity. However, magnesium competes more effectively with the lanthanide if the substrate analogue 3-aminoenolpyruvate 2-phosphate (AEP) is present.The fluorescence of the lanthanide is not readily observed on exciting the terbium-enzyme at 280 nm, indicating the absence of tyrosines or tryptophans in the coordination sphere of the metal. Excitation of terbium using 488 nm radiation from an argon ion laser shows the fluorescence of the metal is enhanced by binding to the enzyme. EDTA and carbonate have similar effects. This suggests carboxyl groups are involved in binding metal at the conformational sites of yeast enolase. Measurements of lifetimes of enzyme-bound terbium in the presence and absence of D₂O indicated three moles of water remained on each of the bound metals, independently of the buffer used. If enzyme-bound terbium is assumed to be nine-coordinate, the metal must bind to six groups from the enzyme. The presence of substrate does not markedly affect the emission spectrum of the bound terbium or the number of water molecules remaining on the metal, but calorimetric measurements show that substrate binds to the terbium enzyme.     

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis.

The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.     

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.     

Kinetic and structural characterization of reversibly inactivated beta-lactamase

The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.     

Investigating the role of metal ions in the catalytic mechanism of the yeast RNA triphosphatase

The Saccharomyces cerevisiae RNA triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5'-terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg2+, Mn2+, and Co2+ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate.     

Demonstration of different metal ion-induced calcineurin conformations using a monoclonal antibody

It has been suggested that calcineurin, a calmodulin-stimulated phosphatase, may exist in different metal ion-dependent conformational states (Pallen, C.J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141). Evidence in favor of this hypothesis comes from studies involving a monoclonal antibody, VA1, which is specific for the small (beta) subunit of calcineurin. This antibody inhibits Ni2+-stimulated but not Mn2+-stimulated phosphatase activity against p-nitrophenyl phosphate and phosphorylase kinase. Inhibition is not due to competition of the antibody with substrate or to interference with metal ion binding to the enzyme. Complex formation between the antibody and calcineurin can be demonstrated either in the presence of Mn2+ or Ni2+ or in the absence of metal ion activators. These results indicate that the active conformational states of calcineurin are metal ion dependent, that the monoclonal antibody VA1 affects the Ni2+-induced conformational change of the enzyme, and that the beta subunit of calcineurin plays a critical role in the expression of Ni2+-stimulated phosphatase activity.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

Competitive inhibition of cathepsin C by guanidinium ions and reexamination of substrate inhibition

Cathepsin C, a lysosomal dipeptidyl aminopeptidase, is competitively and reversibly inhibited by guanidinium ions with a Ki approximately 1.5 mM. Loss of activity is not the result of conformational change, subunit dissociation or altered mobility of the enzyme, but rather reflects a specific binding of guanidinium ions to the active site. The finding that cathepsin C is not inhibited by substrate has allowed the kinetic parameters in the presence of guanidinium ion to be determined. Guanidinium significantly decreases the Km of substrate hydrolysis, without changing Vmax. In a novel application of the transferase reaction, the Km of the nucleophile substrate has been determined (11 mM) and found not to be affected by guanidinium, indicating its inhibition of substrate binding to the S, but not the S', site. Inhibition is suggested to be the result of shielding a negative charge on the enzyme important for interaction with the substrate.     

Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.     

Multiple metal ions drive DNA association by PvuII endonuclease

Restriction enzymes serve as important model systems for understanding the role of metal ions in phosphodiester hydrolysis. To this end, a number of laboratories have reported dramatic differences between the metal ion-dependent and metal ion-independent DNA binding behaviors of these systems. In an effort to illuminate the underlying mechanistic details which give rise to these differences, we have quantitatively dissected these equilibrium behaviors into component association and dissociation rates for the representative PvuII endonuclease and use these data to assess the stoichiometry of metal ion involvement in the binding process. The dependence of PvuII cognate DNA on Ca(II) concentration binding appears to be cooperative, exhibiting half-saturation at 0.6 mM metal ion and yielding an n(H) of 3.5 +/- 0.2 per enzyme homodimer. Using both nitrocellulose filter binding and fluorescence assays, we observe that the cognate DNA dissociation rate (k(-)(1) or k(off)) is very slow (10(-)(3) s(-)(1)) and exhibits a shallow dependence on metal ion concentration. DNA trap cleavage experiments with Mg(II) confirm the general irreversibility of DNA binding relative to cleavage, even at low metal ion concentrations. More dramatically, the association rate (k(1) or k(on)) also appears to be cooperative, increasing more than 100-fold between 0.2 and 10 mM Ca(II), with an optimum value of 2.7 x 10(7) M(-)(1) s (-)(1). Hill analysis of the metal ion dependence of k(on) indicates an n(H) of 3.6 +/- 0.2 per enzyme dimer. This value is consistent with the involvement in DNA association of two metal ions per subunit active site, a result which lends new strength to arguments for two-metal ion mechanisms in restriction enzymes.     

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.     

Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59.The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA.We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.     

Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent.

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.     

Improving the catalytic activity of a thermophilic enzyme at low temperatures

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.     

Binding of 3,4,5,6-tetrahydroxyazepanes to the acid-β-glucosidase active site: implications for pharmacological chaperone design for Gaucher disease

Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-β-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.     

Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits [Orhanovi? S., Pavela-Vrancic M. and Flogel-Mrsi? M. (1994) Acta. Pharm.44, 87-95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg2+ binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analyses. An activation study has indicated that Mg2+ enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn2+ or Mg2+ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release. APase displays higher activity in the presence of Mg2+, as binding of Mg2+ increases the rate of conformational change. A conformationally controlled and Mg2+-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of Pi into the environment.