Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin

Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.     

Probing the mechanism of inosine monophosphate dehydrogenase with kinetic isotope effects and NMR determination of the hydride transfer stereospecificity

The mechanism of human type II inosine monophosphate dehydrogenase has been probed by measurements of primary deuterium kinetic isotope effects, and by determination of the stereochemical course of the reaction. The deuterium isotope effects on Vmax from [2-deutero]-IMP are unity for reactions with a variety of monovalent cation activators (K+, NH4+, Na+, Rb+) of various efficacy. In each case normal effects on Vmax/K(m) in the range of 1.9 to 3.5 are observed for both IMP and NAD, and are larger for NAD. These results demonstrate that both substrates can dissociate from the E.M+.IMP.NAD complex, therefore the kinetic mechanism is not ordered as previous steady-state kinetic studies have suggested. Comparison of reaction rates in D2O and H2O show no 2H isotope effect on Vmax, and a < or = twofold decrease in Vmax/K(m); thus, a proton transfer from solvent is not rate-limiting in turnover. The NMR spectrum of the [4-deutero]NADH produced in the reaction of [2-deutero]-IMP and NAD shows that the hydrogen is transferred to the B, or pro-S, side of the nicotinamide ring. Presteady-state kinetic experiments reveal a burst of NADH formation in the first turnover, demonstrating that a late step in the mechanism is rate-limiting. The rate of the burst phase is reduced approximately twofold with [2-deutero]IMP as substrate, indicating that the hydride transfer step is kinetically significant early in the reaction.     

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.     

Carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase

The carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) was examined using a recombinant enzyme. Kinetic parameters were obtained for 48 carbonyl group-containing substrates, including aromatic aldehydes, aromatic ketones, cycloketones, quinones, aliphatic aldehydes and aliphatic ketones. 20 beta-HSD showed a high affinity towards quinones, such as 9,10-phenanthrenequinone, alpha-naphthoquinone and menadione (Km values of 4, 2 and 5 microM, respectively), and the substrate utilization efficiency (Vmax/Km) of the enzyme against these quinones was very high. Cyclohexanone and 2-methylcyclohexanone were also reduced with a high Vmax/Km value, but not cyclopentanone or 2-methylcyclopentanone. Various aromatic aldehydes and ketones including benzaldehyde- and acetophenone-derivatives were reduced by 20 beta-HSD. Especially, 4-nitrobenzaldehyde and 4-nitroacetophenone were reduced with high Vmax/Km values in the related compounds. The enzyme also reduced the pyridine-derivatives, 2-, 3-, and 4-benzoylpyridine, with the Vmax/Km value for 2-benzoylpyridine being the highest. 20 beta-HSD reduced aliphatic aldehydes and aliphatic ketones, but was more effective on the former. The correlation between the structure of carbonyl compounds and their substrate Vmax/Km is discussed.     

Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.     

Partial functional deficiency of E160D flap endonuclease-1 mutant in vitro and in vivo is due to defective cleavage of DNA substrates

To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene. The mutants were then expressed in Escherichia coli and purified using a His-tag. Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exception of an E160D mutant. This mutant retained wild type-like binding ability, specificity, and partial catalytic activity. Detailed steady state and pre-steady state kinetic analysis revealed that the functional deficiency of this mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.     

Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity

We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.     

Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity

It is thought that only a limited number of residues in the extended binding pocket of coagulation proteases are critical for substrate and inhibitor specificity. A candidate residue from the crystal structures of thrombin and factor Xa (FXa) that may be critical for specificity at the S2 subsite is residue 99. Residue 99 is Tyr in FXa and Thr in activated protein C (APC). To determine the role of residue 99 in S2 specificity, a Gla-domainless mutant of protein C (GDPC) was prepared in which Thr99 was replaced with Tyr of FXa. GDPC T99Y bound Ca2+ and was activated by the thrombin-thrombomodulin complex normally. The T99Y mutant, similar to FXa, hydrolyzed the chromogenic substrates with a Gly at the P2 positions. This mutant was also inhibited by antithrombin (AT) (k2 = 4.2 +/- 0.2 x 10(1) M-1 s-1), and heparin accelerated the reaction >350-fold (k2 = 1.5 +/- 0.1 x 10(4) M-1 s-1). The T99Y mutant, however, did not activate prothrombin but inactivated factor Va approximately 2-fold better than wild type. To try to switch the specificity of FXa, both Tyr99 and Gln192 of FXa were replaced with those of APC in the Gla-domainless factor X (GDFX Y99T/Q192E). This mutant was folded correctly as it bound Ca2+ with a similar affinity as GDFX and was also activated by the Russell's viper venom at similar rate, but it cleaved the chromogenic substrates with a Gly at the P2 positions poorly. The mutant, instead, cleaved the APC-specific chromogenic substrates efficiently. The Y99T/Q192E mutant became resistant to inhibition by AT in the absence of heparin but was inhibited by AT almost normally in the presence of heparin (k2 = 3.4 +/- 0.5 x 10(5) M-1 s-1). The Y99T/Q192E mutant did not inactivate factor Va, and prothrombin activation by this mutant was impaired. These results indicate that 1) residue 99 is critical for enzyme specificity at the S2 subsite, 2) a role for heparin in acceleration of FXa inhibition by AT may involve the S2-P2 modulation, and 3) the exchange of residues 99 and 192 in FXa and APC may switch the enzyme specificity with the chromogenic substrates and inhibitors but not with the natural substrates.     

Metabolic control during preimplantation mammalian development

The stability properties of six natural mutants of the TEM-1 beta-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the N delta of His-164 and the O delta of Asp-179. The properties of the G238S + E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

Probing the mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues

The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst. Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants. Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate. The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation. E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue. Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide. Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct. This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.     

Horse liver alcohol dehydrogenase-catalyzed oxidation of aldehydes: dismutation precedes net production of reduced nicotinamide adenine dinucleotide

The oxidation of aldehydes by horse liver alcohol dehydrogenase (HL-ADH) is more complex than previously recognized. At low enzyme concentrations and/or high aldehyde concentrations, a pronounced lag in the assay progress curve is observed when the reaction is monitored for NADH production at 340 nm. When the progress of the reaction is followed by 1H NMR spectroscopy, rapid dismutation of the aldehyde substrate into the corresponding acid and alcohol is observed during the lag phase. Steady-state production of NADH commences only after aldehyde concentrations drop below 5% of their initial value; thereafter, NADH production occurs with continuous adjustment of the equilibrium between aldehyde, alcohol, NADH, and NAD+. The steady-state NADH production exhibits normal Michaelis-Menten kinetics and is in accord with earlier studies using much higher enzyme concentrations where no lag phase was reported. These results establish that the ability of HL-ADH to oxidize aldehydes is much greater than previously thought. The relationship between aldehyde dismutase and aldehyde dehydrogenase activities of HL-ADH is discussed.     

Diminished heparin binding of a basic fibroblast growth factor mutant is associated with reduced receptor binding, mitogenesis, plasminogen activator induction, and in vitro angiogenesis

Using modeling of heparin-fibroblast growth factor interactions, we replaced four basic residues of basic fibroblast growth factor (FGF-2) with neutral glutamine residues by site-specific mutagenesis to give the mutants K128Q, K138Q, K128Q-K138Q, R129Q, K134Q, and R129Q-K134Q. The FGF mutants were characterized for their receptor and heparin binding affinities, mitogenic and cell proliferation activities, and their ability to induce plasminogen activator (PA) production and in vitro angiogenesis by cultured endothelial cells. Heparin binding properties and biological activities of the three mutants involving R129 and K134 remained essentially unchanged; however, significant changes for three mutants involving K128 and K138 were found. The KD values for heparin binding for K128Q and K138Q mutants were increased about 10-fold, and that for the K128Q-K138Q double mutant was increased by about 100-fold. The mutant K128Q-K138Q required a 10-fold higher concentration of heparin to promote binding to heparan sulfate proteoglycan (HSPG)-deficient CHO cells transfected with fibroblast growth factor receptor-1 (FGFR1) or to induce DNA synthesis in HSPG-deficient myeloid cells transfected with FGFR1. Binding affinities of the mutants to cell surface receptors on BHK-21 cells, however, were similar to that of wild-type FGF-2. In endothelial cell proliferation assays the activities of K128Q and K128Q-K138Q were about 10-fold lower than that of the wild-type protein, whereas the K138Q mutant exhibited wild-type activity. In addition, the K128Q-K138Q mutant displayed a markedly lowered capacity to induce PA activity in cultured endothelial cells and to form capillary-like structures in an in vitro angiogenesis model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis-- X-ray structure analysis of site-directed mutants

We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.     

General approach to analysis of polymorphic short tandem repeat loci

The role of N-glycosylation in the expression, stability, and ligand recognition by the cocaine- and antidepressant-sensitive human norepinephrine transporter (hNET) was assessed in stably and transiently transfected cell lines. The use of hNET-specific antibodies and the membrane-impermeant biotinylating reagent sulfosuccinimidobiotin establishes that treatment of stably transfected LLC-PK1 cells with tunicamycin depletes surface membranes of mature hNET glycoproteins, which is consistent with a failure of less stable, nonglycosylated subunits to replenish surface compartments. To determine whether N-glycosylation plays a direct role in hNET stability, surface expression, and ligand recognition, we mutated the three hNET canonical N-glycosylation sites (hNETN184, 192, 198Q) and transiently expressed the mutant cDNA in parallel with the parental hNET construct in HeLa and COS cells. hNETN184, 192, 198Q protein exhibited increased electrophoretic mobility (approximately 46 kDa), similar to that of enzymatically N-deglycosylated hNET protein, which confirms the use of canonical sites in the second extracellular loop of the transporter. hNETN184, 192, 198Q protein in HeLa and COS extracts was reduced approximately 50% relative to hNET protein in parallel transfections, demonstrated to arise from a reduction in transporter half-life, which is consistent with the proposed role of N-glycosylation in hNET stability. Both HeLa and COS cells transfected with hNETN184, 192, 198Q exhibit a significantly greater reduction in transport activity than can be accounted for by losses in either total or surface NET protein. Furthermore, sensitivity of catecholamine transport to unlabeled substrate and antagonists was unchanged in the mutant, suggesting that residual nonglycosylated surface hNETs execute a key step in the transport cycle after ligand recognition with reduced efficiency.     

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.