Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake. For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.     

Structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p

Fet3p is a multicopper oxidase (MCO) that functions together with the iron permease, Ftr1p, to support high-affinity Fe uptake in yeast. Fet3p is a ferroxidase that, like ceruloplasmin and hephaestin, couples the oxidation of 4 equiv of Fe(II) to the reduction of O2 to 2 H2O. The ferrous iron specificity of this subclass of MCO proteins has not been delineated by rigorous structure-function analysis. Here the crystal structure of Fet3p has been used as a template to identify the amino acid residues that confer this substrate specificity and then to quantify the contributions they make to this specific reactivity by thermodynamic and kinetic analyses. In terms of the Marcus theory of outer-sphere electron transfer, we show here that D283, E185, and D409 in Fet3p provide a Fe(II) binding site that actually favors ferric iron; this site thus reduces the reduction potential of the bound Fe(II) in comparison to that of aqueous ferrous iron, providing a thermodynamically more robust driving force for electron transfer. In addition, E185 and D409 constitute parts of the electron-transfer pathway from the bound Fe(II) to the protein's type 1 Cu(II). This electronic matrix coupling relies on H-bonds from the carboxylate OD2 atom of each residue to the NE2 NH group of the two histidine ligands at the type 1 Cu site. These two acidic residues and this H-bond network appear to distinguish a fungal ferroxidase from a fungal laccase since the specificity that Fet3p has for Fe(II) is completely lost in a Fet3pE185A/D409A mutant. Indeed, this double mutant functions kinetically better as a laccase, albeit a relatively inefficient one.     

Probing the function of the conserved tryptophan in the flexible loop of the Yersinia protein-tyrosine phosphatase.

The involvement of the strictly conserved Trp354 residue in the catalysis of the Yersinia protein tyrosine phosphatase (PTPase) has been investigated by site-directed mutagenesis and kinetic studies. Crystallographic structural data have revealed that Trp354 interacts with the active site Arg409 and is located at one of the hinge positions of the flexible surface loop (WpD loop) which also harbors the general acid/base (Asp356) essential for catalysis [Schubert, H. L., Fauman, E. B., Stuckey, J. A., Dixon, J. E. & Saper, M. A. (1995) Protein Sci. 4, 1904-1913]. Two mutants were constructed and expressed that contained the Trp354-->Phe and Trp354-->Ala substitutions. The K(m) of the W354F and W354A mutants were not significantly different from that of the wild-type. However, a major decrease in the affinity for oxyanions was observed for the mutants, which is consistent with Trp354 playing a role in aligning Arg409 for oxyanion binding. In addition replacement of Trp354 with Phe or Ala caused a decrease in kcat of 200-fold and 480-fold, respectively, and impaired the ability of the mutant enzymes to stabilize the negative charge in the leaving group at the transition state. In fact, the W354F and W354A mutants exhibited catalytic efficiency and leaving group dependency similar to those observed for the general acid-deficient PTPase D356N. These results indicate that Trp354 is an important residue that keeps the WpD loop in a catalytically competent conformation and positions the general acid/base Asp356 in the correct orientation for proton transfer.     

Interactions of granisetron with an agonist-free 5-HT3A receptor model

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.     

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.     

Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae

The Fet3 protein from Saccharomyces cerevisiae is a multicopper oxidase with specificity toward Fe(II) and Cu(I). Fet3p turnover of Fe(II) supports high affinity iron uptake across the yeast plasma membrane, whereas its turnover of Cu(I) contributes to copper resistance in yeast. The structure of Fet3p has been used to identify possible amino acid residues responsible for this protein's reactivity with Cu(I), and structure-function analyses have confirmed this assignment. Fet3p Met(345) is required for the enzyme's reactivity toward Cu(I). Although the Fet3pM345A mutant exhibits wild type spectral and electrochemical behavior, the kinetic constants for Cu(I) turnover and for single-turnover electron transfer from Cu(I) to the enzyme are significantly reduced. The specificity constant with Cu(I) as substrate is reduced by one-fifth, whereas the electron transfer rate from Cu(I) is reduced 50-fold. This mutation has little effect on the reactivity toward Fe(II), indicating that Met(345) contributes specifically to Fet3p reactivity with the cuprous ion. These kinetic defects render the Fet3pM345A unable to support wild type cellular copper resistance, suggesting that there is a finely tuned copper redox balance at the yeast plasma membrane.     

The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact

High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.     

Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae

The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the approximately 13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase: roles of His 54, Asp130, Glu155, Lys185, and Aspl89

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 A resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S(D) bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S(D) bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-S(D) bond of AdoHcy.     

Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis.

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.     

Mutational analysis of C-lobe ligands of human serum transferrin: insights into the mechanism of iron release

Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive; each monoferric hTF has a free energy of binding that is 82% of diferric hTF.     

Iron metabolism in the yeast

Current data concerning transport, storage and utilization of iron in the yeast cells, particularly Saccharomyces cerevisiae are summarized in the paper. It has been marked that iron uptake in the cells provides by high affinity system, it function is carried out by protein complex Fet3-Ftr1, and Fet4, protein with low affinity to iron ion. The both systems utilize Fe(II). Furthermore, the active site of the protein Fre1 is exposed on the outer side of plasmalemma. This protein, due to ferrireductase activity, provides availability of Fe(III) to the cell. The information regards to participation of siderophores and metal-proton plasma membrane exchangers Smf1 in iron transport is brought. Particular attention is given to regulation of expression of the genes, coding the iron metabolic systems. Some aspects of iron utilization for Fe-S-containing enzymes synthesis are lighted. It has been concluded that the yeast is a perspective subject for studying balance of living organisms between iron essentiality and its ability to trigger free radical reactions.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides.

This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Mutational analysis of catecholamine binding in tyrosine hydroxylase

Tyrosine hydroxylase (TH) performs the first and rate-limiting step in the synthesis of catecholamines, which feed back to regulate the enzyme by irreversibly binding to a high-affinity site and inhibiting TH activity. Phosphorylation of Ser40 relieves this inhibition by allowing dissociation of catecholamine. We have recently documented the existence of a low-affinity catecholamine binding which is dissociable, is not abolished by phosphorylation, and inhibits TH by competing with the essential cofactor, tetrahydrobiopterin. Here, we have substituted a number of active site residues to determine the structural nature of the low- and high-affinity sites. E332D and Y371F increased the IC(50) of dopamine for the low-affinity site 10-fold and 7 0-fold, respectively, in phosphorylated TH, indicating dramatic reductions in affinity. Only 2-4-fold increases in IC(50) were measured in the nonphosphorylated forms of E332D and Y371F and also in L294A and F300Y. This suggests that while the magnitude of low-affinity site inhibition in wild-type TH remains the same upon TH phosphorylation as previously shown, the active site structure changes to place greater importance on E332 and Y371. Changes to high affinity binding were also measured, including a loss of competition with tetrahydrobiopterin for E332D, A297L, and Y371F and a decreased ability to inhibit catalysis (V(max)) for A297L and Y371F. The common roles of E332 and Y371 indicate that the low- and high-affinity catecholamine binding sites are colocalized in the active site, but due to simultaneous binding, may exist in separate monomers of the TH tetramer.