Ectodomain Movements of an ATP-gated Ion Channel (P2X2 Receptor) Probed by Disulfide Locking

The ectodomain of the P2X receptor is formed mainly from two- or three-stranded β-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the β-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.     

Direct Gating of ATP-activated Ion Channels (P2X2 Receptors) by Lipophilic Attachment at the Outer End of the Second Transmembrane Domain

The ionic pore of the P2X receptor passes through the central axis of six transmembrane (TM) helices, two from each of three subunits. Val⁴⁸ and Ile³²⁸ are at the outer end of TM1 and TM2, respectively. Homology models of the open and closed states of P2X2 indicate that pore opening is associated with a large lateral displacement of Ile³²⁸. In addition, molecular dynamics simulations suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was activated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-d-glucamine. The ATP-binding residue Lys⁶⁹ was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) receptors. The propyl-MTS did not open P2X2 receptors in which the Val⁴⁸ side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val⁴⁸ and Ile³²⁸ stabilizes the closed channel and that this is broken by covalent attachment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during channel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the channel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the position of the TMs within the bilayer.     

C terminus of the P2X7 receptor: treasure hunting

P2X receptor (P2XR) is a family of the ATP-gated ion channel family and can permeabilize the plasma membrane to small cations such as potassium, sodium, and calcium, resulting in cellular depolarization. There are seven P2XR that have been described and cloned, with 45% identity in amino acid sequence. Each P2X receptors has two transmembrane domains that are separated by an extracellular loop and an intracellular N and C terminus. Unlike the other P2X receptors, the P2X7R has a larger C terminus with an extra 200 amino acid residues compared with the other receptors. The C terminus of the P2X7R has been implicated in regulating receptor function including signaling pathway activation, cellular localization, protein–protein interactions, and post-translational modification (PTM). In the present review, we discuss the role of the P2X7R C terminus in regards to receptor function, describe the specific domains and motifs found therein and compare the C terminus sequence with others proteins to discover predicted domains or sites of PTM.     

Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC₅₀ for H33C/S345C before dithiothreitol treatment was ∼2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC₅₀ to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

Alternative Splicing of the N-Terminal Cytosolic and Transmembrane Domains of P2X7 Controls Gating of the Ion Channel by ADP-Ribosylation

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.     

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.     

Growth-active peptides are produced from alpha-lactalbumin and lysozyme

We determined the growth-active domains of milk-growth factor (MGF), human alpha-lactalbumin (HMLA) and human lysozyme (HMLZ), and their sequences. Fetal calf serum (FCS) showed inhibitors against proteases. The growth-stimulation of IMR90 cells in CG medium (free-serum) without FCS was induced in a dose-dependent manner up to 400 ng/ml of HMLA, HMLZ or chicken lysozyme (ChLZ), and also in a time-dependent manner until 48 h but was induced gradually until 1000 ng/ml of bovine alpha-lactalbumin (BVLA). The HMLAL6-peptide (HMLAL6), a cleaved product from HMLA by Endpeptidase Lys C, was growth-stimulative. The sequence of HMLAL6 was matched to 35 amino-acid residues (from No. 59 to No. 93 of HMLA), owing to the sequences of HMLAL6R3, HMLAL6R5 and HMLAL6R7 after the reduction of HMLAL6. The sequences of the reduced peptides from MGFL7-peptide (MGFL7: a cleaved product from MGF by Endpeptidase lysine C matched to those of the peptides from HMLAL6, and were similarly identified as the partial sequence of HMLA (59-93, H(2)N-L.W.C.?.K./S.S.Q.V.P.Q.S.R.N.I.?.D.I.S.?.D.K./F.L. D.D.D.I.T.D.D.I.M.?.A.-COOH). The sequence of HMLZ is similar to that of HMLA. HMLZT7-peptide (HMLZT7), a cleaved product of HMLZ by trypsin, was confirmed to have growth-stimulating activity and it's sequence was partially identified as Y. W.?.N.D.G.K.T.P.G.A.V.N.A.?.H.L. -, owing to the results of HMLZT7R1 (reduction of HMLZT7) and HMLZA7R2 (reduction of HMLZA7-peptide (HMLZA7) cleaved product of HMLZ by Endpeptidase Arg C) and is accordingly the sequence from No. 63 to No. 97 of HMLZ. Therefore, the peptides produced from LA and LZ by proteolysis may play a role of growth-stimulation.     

An investigation of cysteine mutants on the cytoplasmic loop X/XI in the melibiose transporter of Escherichia coli by using thiol reagents: implication of structural conservation of charged residues

The melibiose transporter (Mel B) of Escherichia coli is a cation-coupled (H(+), Li(+), and Na(+)) membrane protein (MW 50 kDa) consisting of 12 transmembrane helices that are connected by periplasmic and cytoplasmic loops, with both the C- and N-ends located on the cytoplasmic side of the membrane. Previous investigations on the largest cytoplasmic loop X/XI indicated that it is a functional re-entrant loop. In this communication, the cysteine mutants on loop X/XI were studied with charged thiol reagents MTSES, MTSET, and IAA for both the inhibition patterns and charge replacement/function rescue of inactive mutants in which the original charged residues were replaced by neutral cysteines. Strong inhibitions were observed in T373C and V376C by both MTSES and MTSET, consistent with previous results of PCMBS inhibition. The thiol reagents failed to recover the activities of inactive mutants D351C, D354C, and R363C and to inhibit active mutants E357C, K359C, and E365C to any significant extent, suggesting a structural conservation at D351, D354, and R363 and tolerance of structural variations at E357, K359, and E365. The results are consistent with previous observation of structural conservation of functionally charged residues in the transmembrane domains and extend to a loop the contention that in the melibiose transporter functionally important charged residues are structurally conserved.     

Regions of the amino terminus of the P2X1 receptor required for modification by phorbol ester and mGluR1α receptors

The potentiation of P2X₁ receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1α receptors was sensitive to inhibition of novel forms of protein kinase C. Potentiation was also reduced by co-expression of an amino terminal P2X₁ receptor minigene. Cysteine point mutants of residues Tyr¹⁶-Gly³⁰ were expressed in Xenopus oocytes. Peak current amplitudes to ATP for Y16C, T18C and R20C mutants were reduced, however this did not result from a decrease in surface expression of the channels. The majority of the mutants showed changes in the time-course of desensitization of ATP evoked currents indicating the important role of this region in regulation of channel properties. PMA and mGluR1α potentiation was abolished for the mutants Y16C, T18C, R20C, K27C and G30C. Minigenes incorporating either Y16C, K27C, V29C or G30C still inhibited PMA responses. However D17C, T18C or R20C mutant minigenes were no longer effective suggesting that these residues are important for interaction with regulatory factors. These results demonstrate that the conserved YXTXK/R sequence and a region with a conserved glycine residue close to the first transmembrane segment contribute to PMA and GPCR regulation of P2X₁ receptors.     

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.     

Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors

The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and αβ-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.     

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.     

The alpha 1 (VIII) collagen gene is homologous to the alpha 1 (X) collagen gene and contains a large exon encoding the entire triple helical and carboxyl-terminal non-triple helical domains of the alpha 1 (VIII) polypeptide

We recently cloned and sequenced alpha 1 (VIII) collagen cDNAs and demonstrated that type VIII collagen is a short-chain collagen that contains both triple helical and carboxyl-terminal non-triple helical domains similar to those of type X collagen (Yamaguchi, N., Benya, P., van der Rest, M., and Ninomiya, Y. (1989) J. Biol. Chem. 264, 16022-16029). We report here on the structural organization of the gene encoding the rabbit alpha 1 (VIII) collagen chain. The alpha 1 (VIII) gene contains four exons, whose sizes are 69, 120, 331, and 2278 base pairs. The first and second exons encode only 5'-untranslated sequences, whereas the third exon codes for a very short (3 nucleotides) stretch of 5'-untranslated sequence, the signal peptide, and almost the entire amino-terminal non-triple helical (NC2) domain (109 1/3 codons). Interestingly, the last exon encodes the rest of the translated region, including 7 2/3 codons of the NC2 domains, the complete triple helical domain (COL1, 454 amino acid residues), the entire carboxyl-terminal non-triple helical domain (NC1, 173 amino acid residues), and the 3'-untranslated region. This exon-intron structure is in stark contrast to the multi-exon structure of the fibrillar collagen (types I, II, III, V, and XI) genes, but it is remarkably similar to that of the type X collagen gene (LuValle, P., Ninomiya, Y., Rosenblum, N. D., and Olsen, B. R. (1988) J. Biol. Chem. 263, 18278-18385). The data suggest that the alpha 1 (VIII) and the alpha 1 (X) genes belong to the same subclass within the collagen family and that they arose from a common evolutionary precursor.     

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.     

Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor

The first hydrophobic segment of the rat P2X(2) receptor extends from residue Leu(29) to Val(51). In the rat P2X(2) receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp(15) through Thr(60)) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 microM). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as alphabeta-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val(48) at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

The role of positively charged amino acids in ATP recognition by human P2X(1) receptors

P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.