Role of conserved tyrosine 343 in intramolecular electron transfer in human sulfite oxidase

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.     

Alteration of specific activity and stability of thermostable neutral protease by site-directed mutagenesis.

On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis. When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared. F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme. When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.     

Alteration of specific activity and stability of thermostable neutral protease by site-directed mutagenesis.

On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis. When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared. F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme. When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.     

Switching kinetic mechanism and putative proton donor by directed mutagenesis of glutathione reductase

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase, Tyr-177 (the residue corresponding to Tyr-197 in the NADPH-binding pocket of the homologous human enzyme) was changed to phenylalanine (Y177F), serine (Y177S), and glycine (Y177G). The catalytic activity of the Y177F mutant was very similar to that of the wild-type enzyme, but that of the Y177S and Y177G mutants was substantially diminished. However, all three mutants retained the ability to protect the reduced flavin from adventitious oxidation, indicating that Tyr-177 does not act as a simple "lid" on the NADPH-binding pocket and that the protection of the reduced enzyme must be due largely to burial of the isoalloxazine ring in the protein. The wild-type enzyme and Y177F mutant displayed ping-pong kinetics, but the Y177S and Y177G mutants appeared to have switched to an ordered sequential mechanism. This could be explained by supposing that the enzyme normally functions by a hybrid kinetic mechanism and that the Y177S and Y177G mutations diverted flux from the ping-pong loop favored by the wild-type enzyme to an ordered sequential loop. The necessary change in the partitioning of the common E-NADPH intermediate could be caused by a slowing of the formation of the EH2 intermediate on the ping-pong loop, or by the observed concomitant fall in the Km for glutathione favoring flux through the ordered sequential loop. In another experiment, His-439, thought to act as a proton donor/acceptor in the glutathione-binding pocket, was mutated to a glutamine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.     

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

An active site tyrosine influences the ability of the dimethyl sulfoxide reductase family of molybdopterin enzymes to reduce S-oxides

Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilize S-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me(3)NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me(3)NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.     

Mechanistic roles of Ser-114, Tyr-155, and Lys-159 in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a short chain dehydrogenase/reductase, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. A catalytic triad of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed based on structural analysis and sequence alignment of the short chain dehydrogenase/reductase family. The 3alpha-HSD/CR-catalyzed reaction has not been kinetically analyzed in detail, however. In this study, we combined steady-state kinetics, site-directed mutagenesis, and pH profile to explore the function of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR-catalyzed reaction. The catalytic efficiency of wild-type and mutants S114A, Y155F, K159A, and Y155F/K159A is 4.3 x 10(7), 7.3 x 10(4), 1.7 x 10(4), 2.4 x 10(5), and 71 m(-1)s(-1), respectively. The values of pKa on kcat/Km for the wild-type, S114A, Y155F, K159A, and Y155F/K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively. Mutant S114A/Y155F exhibits a pH-independent profile with 10(-5) times of wild-type activity at pH 10.5. The activity decreases as the pH lowers, which indicates that a functional group with an apparent pKa of 7.2 is involved in the general base catalysis for wild-type 3alpha-HSD/CR. The pKa shift to 9.1 for mutant K159A suggests the role of Lys-159 is to lower the pKa of the residues involved in the general base catalysis. Because pH dependence is observed for both S114A and Y155F mutants and pH independence is observed in S114A/Y155F, Tyr-155 may be important as a general base catalysis in the wild-type, whereas Ser-114 may act as a general base on mutant Y155F to catalyze the reaction.     

Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of FAD reduction and role of active site residues His-225 and Tyr-259 in Arthrobacter globiformis dimethylglycine oxidase: analysis of mutant structure and catalytic function

Residues His-225 and Tyr-259 are located close to the FAD in the dehydrogenase active site of the bifunctional dimethylglycine oxidase (DMGO) of Arthrobacter globiformis. We have suggested [Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038-4048] that these residues are involved in abstraction of a proton from the substrate amine group of dimethylglycine prior to C-H bond breakage and FAD reduction. To investigate this proposal, we have isolated two mutant forms of DMGO in which (i) His-225 is replaced with Gln-225 (H225Q mutant) and (ii) Tyr-259 is replaced with Phe-259 (Y259F mutant). Both mutant enzymes retain the ability to oxidize substrate, but the steady-state turnover of the Y259F mutant is attenuated more than 200-fold. Only modest changes in kinetic parameters are observed for the H225Q mutant during steady-state turnover. Stopped-flow studies indicate that the rate of FAD reduction in the Y259F enzyme is substantially impaired by a factor of approximately 1500 compared with that of the wild-type enzyme, suggesting a key role for this residue in the reductive half-reaction of the enzyme. The kinetics of FAD reduction in the H225Q enzyme are complex and involve three discrete kinetic phases that are attributed to different conformational states of this mutant, evidence for which is provided by crystallographic analysis. Neither the H225Q enzyme nor the Y259F enzyme stabilizes the FADH(2)-iminium charge-transfer complex observed previously in stopped-flow studies with the wild-type enzyme. Our studies are consistent with a key role for Tyr-259, but not His-225, in deprotonation of the substrate amine group prior to FAD reduction. We infer that residue His-225 is likely to modulate the acid-base properties of Tyr-259 by perturbing the pK(a) of Tyr-259 and thus fine-tunes the reaction chemistry to facilitate proton abstraction under physiological conditions. Our data are discussed in the context of the crystallographic data for DMGO and also in relation to contemporary mechanisms for flavoprotein-catalyzed oxidation of amine substrates.     

Formation in vitro of hybrid dimers of H463F and Y74F mutant Escherichia coli tryptophan indole-lyase rescues activity with L-tryptophan

Y74F and H463F mutant forms of Escherichia coli tryptophan indole-lyase (Trpase) have been prepared. These mutant proteins have very low activity with L-Trp as substrate (kcat and kcat/Km values less than 0.1% of wild-type Trpase). In contrast, these mutant enzymes exhibit much higher activity with S-(o-nitrophenyl)-L-cysteine and S-ethyl-L-cysteine (kcat/Km values about 1-50% of wild-type Trpase). Thus, Tyr-74 and His-463 are important for the substrate specificity of Trpase for L-Trp. H463F Trpase is not inhibited by a potent inhibitor of wild-type Trpase, oxindolyl-L-alanine, and does not exhibit the pK(a) of 6.0 seen in previous pH dependence studies [Kiick, D. M., and Phillips, R. S. (1988) Biochemistry 27, 7333]. These results suggest that His-463 may be the catalytic base with a pK(a) of 6.0 and Tyr-74 may be a general acid catalyst for the elimination step, as we found previously with tyrosine phenol-lyase [Chen, H., Demidkina, T. V., and Phillips, R. S. (1995) Biochemistry 34, 12776]. H463F Trpase reacts with L-Trp and S-ethyl-L-cysteine in rapid-scanning stopped-flow experiments to form equilibrating mixtures of external aldimine and quinonoid intermediates, similar to those observed with wild-type Trpase. In contrast to the results with wild-type Trpase, the addition of benzimidazole to reactions of H463F Trpase with L-Trp does not result in the formation of an aminoacrylate intermediate. However, addition of benzimidazole with S-ethyl-L-cysteine results in the formation of an aminoacrylate intermediate, with lambda(max) at 345 nm, as was seen previously with wild-type Trpase [Phillips, R. S. (1991) Biochemistry 30, 5927]. This suggests that His-463 plays a specific role in the elimination step of the reaction of L-Trp. Refolding of equimolar mixtures of H463F and Y74F Trpase after unfolding in 4 M guanidine hydrochloride results in a dramatic increase in activity with L-Trp, indicating the formation of a hybrid H463F/Y74F dimer with one normal active site.     

The role of Tyr-169 of trimethylamine dehydrogenase in substrate oxidation and magnetic interaction between FMN cofactor and the 4Fe/4S center

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected in klim/Kd), and in the breakdown of the Eox.S complex (as reflected in klim). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pKa approximately 9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.     

The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme

To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wild-type enzyme. (1)H-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.     

Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 --> F mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.     

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression

Androgen receptor (AR) signaling plays important roles in breast cancer progression. We show here that Kindlin-2, a focal adhesion protein, is critically involved in the promotion of AR signaling and breast cancer progression. Kindlin-2 physically associates with AR and Src through its two neighboring domains, namely F1 and F0 domains, resulting in formation of a Kindlin-2-AR-Src supramolecular complex and consequently facilitating Src-mediated AR Tyr-534 phosphorylation and signaling. Depletion of Kindlin-2 was sufficient to suppress Src-mediated AR Tyr-534 phosphorylation and signaling, resulting in diminished breast cancer cell proliferation and migration. Re-expression of wild-type Kindlin-2, but not AR-binding-defective or Src-binding-defective mutant forms of Kindlin-2, in Kindlin-2-deficient cells restored AR Tyr-534 phosphorylation, signaling, breast cancer cell proliferation and migration. Furthermore, re-introduction of phosphor-mimic mutant AR-Y534D, but not wild-type AR reversed Kindlin-2 deficiency-induced inhibition of AR signaling and breast cancer progression. Finally, using a genetic knockout strategy, we show that ablation of Kindlin-2 from mammary tumors in mouse significantly reduced AR Tyr-534 phosphorylation, breast tumor progression and metastasis in vivo. Our results suggest a critical role of Kindlin-2 in promoting breast cancer progression and shed light on the molecular mechanism through which it functions in this process.Subject terms: Cell signalling, Breast cancer     

Studies on the reaction mechanism of Rhodotorula gracilis D-amino-acid oxidase. Role of the highly conserved Tyr-223 on substrate binding and catalysis

We have studied D-amino-acid oxidase from Rhodotorula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis. Tyr-223, one of the very few conserved residues among D-amino-acid oxidases, has been mutated to phenylalanine and to serine. Both mutants are active catalysts in turnover with D-alanine, and they are reduced by D-alanine slightly faster than wild-type enzyme. The Tyr-223 --> Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 --> Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active-site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis.     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.     

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.