Cloning and sequencing of phenylethylamine oxidase from Arthrobacter globiformis and implication of Tyr-382 as the precursor to its covalently bound quinone cofactor

The gene of Arthrobacter globiformis encoding a quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues is a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which has been found to be highly conserved in other copper amine oxidase. Mutation of the former Tyr (Tyr-382) of the recombinant enzyme into Phe resulted in the complete loss of catalytic activity and disappearance of the quinone compound that is specifically detected in the wild-type enzyme, suggesting that Tyr-382 is the precursor to the covalently-bound cofactor, most probably topa quinone. Furthermore, the expression of the active, quinone-containing enzyme in Escherichia coli cells was markedly dependent on the presence of Cu2+ ions in the culture medium, and the inactive, Cu2(+)-deficient enzyme produced without Cu2+ ions could be converted to the active quinone form by reconstitution with Cu2+ ions.     

Cloning of the maoA gene that encodes aromatic amine oxidase of Escherichia coli W3350 and characterization of the overexpressed enzyme

The mao operon of Escherichia coli W3350, which comprises the genes maoC and maoA, was cloned and appeared to be similar to that of Klebsiella aerogenes [Sugino, H., Sasaki, M., Azakami, H., Yamashita, M. & Murooka, Y. (1992) J. Bacteriol. 174, 2485-2492]. The gene that encodes aromatic amine oxidase (maoA) was isolated, sequenced, and expressed in E. coli TG2. The purified enzyme exhibited properties characteristic of a copper/topaquinone(TPQ)-containing amine oxidase with respect to the optical absorption and EPR spectra, the size of the subunits, and the optical absorption spectra obtained upon derivatization with hydrazines. However, high-resolution anion-exchange chromatography revealed that the preparation was heterogeneous. The enzyme preparation appeared to consist of at least four enzyme species with different specific activities, A474nm/A340nm ratios and TPQ/subunit ratios. Since the overall properties of the overexpressed enzyme and the authentic enzyme were similar and the separated enzyme species had identical N-terminal amino acid sequences, the heterogeneity does not seem to be caused by improper expression of the gene in the recombinant strain but by factors that interfere with the processing of the specific tyrosine in the precursor enzyme to functional TPQ. Although other causes cannot be excluded, the spectral data and TPQ/subunit ratios reported in the literature for other amine oxidases suggest that suboptimal synthesis of functional TPQ also occurs in other organisms.     

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase

Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.     

Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.     

Function-structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase

Rat hepatic squalene synthase (RSS, EC 2.5.1.21) contains three conserved sections, A, B, and C, that were proposed to be involved in catalysis (McKenzie, T. L., Jiang, G., Straubhaar, J. R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374). Here we use the high expression vector pTrxRSS and site-directed mutagenesis to determine the specific residues in these sections that are essential for the two reactions catalyzed by RSS. Section C mutants F288Y, F288L, F286Y, F286W, F286L, Q293N, and Q283E accumulate presqualene diphosphate (PSPP) from trans-farnesyl diphosphate (FPP) with reduced production of squalene. F288L, which retains approximately 50% first step activity, displays only residual activity (0.2%) in the production of squalene from either FPP or PSPP. Substitution of either Phe288 or Phe286 with charged residues completely abolishes the enzyme activity. Thus, F288W, F288D, F288R, F286D, and F286R cannot produce squalene from either FPP or PSPP. All single residue mutants in Section A, except Tyr171, retain most of the RSS activity, with no detectable accumulation of PSPP in an assay mixture complete with NADPH. Y171F, Y171S, and Y171W are all inactive. Section B, which binds the diphosphate moieties of the allylic diphosphate subtrates, contains four negatively charged residues: Glu222, Glu226, Asp219, and Asp223. The two Glu residues can be replaced with neutral or with positively charged residues without signficantly affecting enzyme activity. However, replacement of either Asp residues with Asn eliminates all but a residual level of activity, and substitution with Glu abolishes all activity. These results indicate that 1) Section C, in particular Phe288, may be involved in the second step of catalysis, 2) Tyr171 of Section A is essential for catalysis, most likely for the first reaction, 3) the two Asp residues in Section B are essential for the activity and most likely bind the substrate via magnesium salt bridges. Based on these results, a mechanism for the first reaction is proposed.     

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.     

Relationship between conserved consensus site residues and the productive conformation for the TPQ cofactor in a copper-containing amine oxidase from yeast

A highly conserved asparagine residue is contained in the consensus site sequences of all known copper-containing amine oxidases (CAOs). On the basis of published crystallographic structures, the asparagine is found to reside proximal to the active site redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ). In this study, the conserved asparagine was changed to an alanine in a CAO from Hansenula polymorpha expressed in Saccharomyces cerevisiae, and the mutant's catalytic properties were characterized using steady-state kinetics and resonance Raman spectroscopy. Several lines of evidence point to TPQ exisiting in an nonproductive orientation in the mutant, including reductions in several steady-state parameters and an accumulation of an inactive product Schiff base complex when the enzyme is incubated with methylamine as the substrate. This product Schiff base complex was previously found to form following mutation of another conserved consensus site residue, a glutamate (or aspartate) at the C + 1 position from TPQ [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478]. The results suggest that these two residues are crucial in maintaining the balance of cofactor mobility versus rigidity expected to be necessary during the dual processes of biogenesis and catalysis, respectively, that all CAOs must accomplish. In addition, a previously unidentified structural linkage between these two highly conserved residues is proposed which spans both subunits of the dimeric CAOs, and may have implications for intersubunit communication.     

Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.     

Glutamate-459 is important for Escherichia coli branching enzyme activity

The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan. The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site. By primary structure analysis, a conserved position unique to branching enzymes has been identified. This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering). Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action. We used site-directed mutagenesis of the Glu-459 residue in the E. coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes. A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme. Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.     

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.     

The yeast mic2 mutant is defective in the formation of mannosyl-diinositolphosphorylceramide

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243-246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp74, Asp200, Asp318 and Glu381 are located on the putative cytoplasmic side, and Asp39 and Glu345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu345 and Glu381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp318 is located in cytoplasmic loop 10-11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2-3 [Yamaguchi et al., Biochemistry 31 (1992) 8344-8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.     

Role of beta87 Thr in the beta6 Val acceptor site during deoxy Hb S polymerization

Three new Hb S variants containing beta87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of the beta87 position in stability and polymerization of deoxy Hb S. Previous studies showed that hydrophobicity at beta85 Phe and beta88 Leu is critical for stabilization of hemoglobin. Results with the three Hb S beta87 variants, however, showed minimal differences in stability, suggesting that beta87 amino acid hydrophobicity is not critical for stabilization of hemoglobin. Polymerization properties of the variants in the deoxy form, however, were affected by the beta87 amino acid. Polymerization of Hb S beta87 Thr --> Leu and Hb S beta87 Thr --> Trp was preceded by a delay time like Hb S, while Hb S beta87 Thr --> Asp did not show a delay time. In addition, changes in time required for half polymer formation (T1/2) as a function of hemoglobin concentration for Hb S beta87 Thr --> Asp were similar to that for beta87 Thr --> Gln. Hb S beta87 Thr --> Leu polymerized at a lower hemoglobin concentration than Hb S while beta87 Thr --> Trp and Hb S beta87 Thr --> Asp required much higher hemoglobin concentrations for polymer formation. Critical concentration required for deoxy Hb S beta87 Thr --> Asp polymerization was 6- and 2.3-fold greater than that for Hb S beta85 Phe --> Glu and Hb S beta88 Leu --> Glu, respectively. These results suggest that even though beta87 Thr is not a direct interaction site for beta6 Val in deoxy Hb S polymers, it does play a critical role in formation of the hydrophobic acceptor pocket which then promotes protein-protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S.     

Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.     

Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics

Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed. Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate. Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.     

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.     

Resonance raman characterization of reaction centers with an Asp residue near the photoactive bacteriopheophytin

Qy-excitation resonance Raman (RR) studies are reported for a series of Rhodobacter capsulatus reaction centers (RCs) containing mutations at L-polypeptide residue 121 near the photoactive bacteriopheophytin (BPhL). The studies focus on the electronic/structural perturbations of BPhL induced by replacing the native Phe with an Asp residue. Earlier work has shown that the electron-transfer properties of F(L121)D RCs are closely related to those of RCs in which BPhL is replaced by bacteriochlorophyll (BChl) (beta-type RCs) or by pheophytin. In addition to the F(L121)D single mutant, RR studies were performed on the F(L121)D/E(L104)L double mutant, which additionally removes the hydrogen bond between BPhL and the native Glu L104 residue. The vibrational signatures of BPhL in the single and double mutants containing Asp L121 are compared with one another and with those of BPhL in both wild-type and F(L121)L RCs. The replacement of the aromatic Phe residue with Leu has no discernible effect on the vibrational properties of BPhL, a finding in concert with the previously reported absence of an effect of the mutation on the electron-transfer characteristics of the RC. In contrast, replacement of Phe with Asp significantly perturbs the vibrational characteristics of BPhL, and in a manner most consistent with Asp L121 being deprotonated and negatively charged. The negative charge of the carboxyl group of Asp L121 interacts with the pi-electron system of BPhL in a relatively nonspecific fashion, diminishing the contribution of charge-separated resonance forms of the C9-keto group to the electronic structure of the cofactor. The presence of a negative charge near BPhL is consistent with the known photochemistry of F(L121)D RCs, which indicates that the free energy of P+BPhL- is substantially higher than in wild-type RCs.     

Readability of self-report clinical outcome measures

Since plasma is generally employed for amino acid analysis, we compared amino acid levels in plasma with those in serum for healthy individuals and examined the influence of separation and storage conditions on the stability of the samples. Then, we determined the amino acid levels of frozen serum samples obtained from sarin poisoned patients. A. Comparison of Amino Acid Levels in Plasma and Those in Serum Blood was collected from 5 healthy individuals. Then, heparinated plasma and serum were separated by centrifugation immediately after blood collection. Serum was also separated by centrifugation after standing whole blood at room temperature for 1 hour. Frozen plasma and serum were store at -40 degrees C for 5 months. All were subjected to analysis in an amino acid analyzer. It was found that the cystine (Cys) and 3-methyl-histidine (3-M-His) levels in serum and plasma were affected when stored in a frozen state, that the aspartate (Asp) level was changed according to the method of collecting serum, and that the taurine (Tau) and ornithine (Orn) levels were affected by standing blood. B. Analysis of Blood Taken from Sarin Poisoned Patients Twelve sarin poisoned patients were selected as subjects, and serum cholinesterase (Ch-E) and serum albumin (Alb) levels were determined. Amino acid analysis was conducted using an amino acid analyzer. Serum samples which had been obtained from the 6 patients and frozen and stored at -40 degrees C from 5 months were used for amino acid analysis. As a result, the serum Ch-E level decreased and the Alb level tended to rise. Since the Ch-E/Alb ratio was reduced in the sarin poisoned patients, it is considered useful for discrimination from liver cirrhosis in which both Ch-E and Alb levels decreased. Amino acid levels in the serum obtained from the sarin poisoned patients were compared with those of healthy individuals, both of which had been stored under the same conditions. There were significant differences in Asp, glutamate (Glu), phenylalanine (Phe), 3-M-His, glutamine (Gln), and Cys levels. The Glu, Phe, and Gln levels were not affected by storage of serum in a frozen state, while the Glu and Phe levels were elevated and the Gln level was reduced. Although Cys exhibited lower values in frozen serum samples, the Cys level was elevated with a rise in the serum Ch-E levels. Therefore, we deduced that Cys metabolism disorders also occur in sarin poisoning. As stated above, the Glu and Phe levels were elevated and the Gln and Cys levels were reduced, suggesting the presence of abnormal amino acid metabolism, in patients with sarin-poisoning.     

Role of tyrosine 129 in the active site of spinach glycolate oxidase

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.