Determining Substrate Selectivity Research Articles

Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection.

Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of ThAdo, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.

Crystal Structure and Mutagenesis of an XYP Subfamily Cyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity and Substrate Specificity.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of SsBcmA from Streptomyces sapporonensis. Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of SsBcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Selective localization of Mfn2 near PINK1 enables its preferential ubiquitination by Parkin on mitochondria.

Mutations in Parkin and PINK1 cause early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: (i) thioester intermediate formation on Parkin and (ii) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyses ubiquitin transfer to substrates. We found that His433 in the RING2 domain contributes to the catalysis of acyl transfer. In cells, the mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin (pUb) in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via pUb at these sites.

Insights into Functional Dimerization of CYP121A1 of Mycobacterium tuberculosis

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. Out of twenty cytochrome P450 enzymes encoded by the Mtb genome, cytochrome P450 121A1 (CYP121A1) is essential, and therefore remains a target of drug design efforts. CYP121A1 mediates a phenol coupling reaction of the tyrosine dipeptide cyclo-L-Tyr-L-Tyr (cYY). In this work, a structure and function investigation of dimerization was performed as an overlooked feature of CYP121A1 function. This investigation showed that CYP121A1 dimers form via intermolecular contacts on the distal surface and are mediated by a network of solvent-exposed hydrophobic residues. Dimerization of CYP121A1 contributes to specific substrate binding in vitro, with 75% loss of function when dimers are disrupted. 19F labeling and nuclear magnetic resonance of the enzyme FG-loop was also combined with protein docking to develop a working model of a functional CYP121A1 dimer. Preliminary crystallographic data of a CYP121A1 homodimer confirm the distal-to-distal orientation of the dimers. These findings suggest a novel role for CYP dimer formation as a contributing factor in determining substrate selectivity, while also providing important mechanistic insight regarding a relevant drug target in the development of novel anti-tuberculosis agents.

Nucleoside Reverse Transcriptase Inhibitor Interaction with Human Equilibrative Nucleoside Transporters 1 and 2

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

A variable loop involved in the substrate selectivity of pinoresinol/lariciresinol reductase from Camellia sinensis

Pinoresinol/lariciresinol reductase (PLR), an NADPH-dependent reductase that catalyzes the sequential reduction of pinoresinol into secoisolariciresinol via Lariciresinol, can lead to the structural and stereochemical diversity of lignans. The relationship between substrate-selective reaction of PLR and sequence homology still remains unclear. In this study, we focused on the contribution of the variable region between PLRs in determining substrate selectivity. Here, two CsPLRs (CsPLR1 and CsPLR2) were identified in the tea plant (Camellia sinensis var. sinensis cv. Shuchazao). In vitro enzymatic assays showed that CsPLR1 could convert (+)- and (−)-pinoresinol into lariciresinol or secoisolariciresinol, whereas CsPLR2 catalyzed (+)-pinoresinol enantioselectively into (−)-secoisolariciresinol. Homology modeling and site-directed mutagenesis were used to examine the role of a variable loop in catalysis and substrate selectivity. The L174I mutant in CsPLR1 lost the capacity to reduce either (+)- or (−)-pinoresinol but retained the ability to catalyze the reduction of (−)-lariciresinol. These findings provide a basis for better understanding of the substrate-selective reaction of PLR.

An Externally Accessible Linker Region in the Sodium-Coupled Phosphate Transporter PiT-1 (SLC20A1) is Important for Transport Function

Background/Aims: Members of the SLC20 cotransporter family (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform housekeeping functions for intracellular P_i homeostasis as well as being implicated in vascular calcification and renal P_i reabsorption. The aims of this study were to investigate the topology of a linker region in PiT-1 between the predicted 2^nd and 3^rd transmembrane domains and to investigate the functional consequences of cysteine substitutions in this region. Methods: Cysteines were substituted at 18 sites in the Xenopus PiT-1 isoform and the mutants were expressed in Xenopus laevis oocytes. Transport function of the mutants was investigated by ³²P tracer or two electrode voltage clamp before and after thiol modification of the novel Cys. Results: Exposure to the thiol reactive reagent resulted in diminished transport function for 7 mutants. The apparent accessibility of 5 of the mutated sites, estimated from the rate of functional thiol modification, was site-dependent. Cysteine substitution at some sites also altered the apparent affinity for P_i and cation (Na⁺/Li⁺) and substrate (phosphate/arsenate) selectivity, further underscoring the importance of this linker in defining PiT-1 transport characteristics. Conclusions: The external accessibility of a linker in PiT-1 was confirmed and sites were identified that determine substrate selectivity and transport function.

Combinatorial Discovery of Peptide Dendrimer Enzyme Models Hydrolyzing Isobutyryl Fluorescein

Two 6750-membered one-bead-one-compound peptide dendrimer combinatorial libraries L (X(4))(8)(LysX(3))(4)(LysX(2))(2)LysX(1) (X(1-4) = 14 different amino acids or deletion, Lys = branching lysine residue) and AcL (with N-terminal acetylation) were prepared by split-and-mix solid phase peptide synthesis. Screening toward fluorogenic substrates for esterase and aldolase activities using the in silica off-bead assay (N. Maillard et al., J. Comb. Chem. 2009, 11, 667-675) and bead decoding by amino acid analysis revealed histidine containing sequences active against fluorescein diacetate. Isobutyryl fluorescein, a related hydrophobic fluorogenic substrate, was preferentially hydrolyzed by dendrimers from library AcL containing hydrophobic residues such as AcH3 (AcHis)(8)(LysLeu)(4)(LysVal)(2)LysLysOH, compared to simple oligohistidine peptides as reference catalysts. Polycationic dendrimers from library L with multiple free N-termini such as H8 (His)(8)(LysβAla)(4)(LysThr)(2)LysaProNH(2) (aPro = (2S,4S)-4-aminoproline) showed stronger reactivity toward 8-acetoxypyrene-1,3,6-trisulfonate with partial acylation of N-termini. These experiments highlight the role of noncatalytic amino acids to determine substrate selectivity in peptide dendrimer esterase models.

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

Glutamate transport is coupled to the co-transport of 3Na(+) and 1H(+) and the countertransport of 1 K(+). However, the mechanism of how this process occurs is not well understood. The crystal structure of an archaeal homolog of the human glutamate transporters, Glt(Ph), has provided the framework to begin to understand the mechanism of transport. The glutamate transporter EAAT2 is different from other subtypes in two respects. First, Li(+) cannot support transport by EAAT2, whereas it can support transport by the other excitatory amino acid transporters, and second, EAAT2 is sensitive to a wider range of blockers than other subtypes. We have investigated the relationship between the cation driving transport and whether the glutamate analogues, l-anti-endo-3,4-methanopyrrolidine-dicarboxylic acid (MPDC) and (2S,4R)-4-methylglutamate (4MG), are substrates or blockers of transport. We have also investigated the molecular basis for these differences. EAAT2 has a Ser residue at position 441 with hairpin loop 2, whereas the corresponding residue in EAAT1 is a Gly residue. We demonstrate that if the transporter has a Ser residue at this position, then 4MG and MPDC are poor substrates in Na(+), and Li(+) cannot support transport of any substrate. Conversely, if the transporter has a Gly residue at this position, then in Na(+) 4MG and MPDC are substrates with efficacy comparable with glutamate, but in Li(+) 4MG and MPDC are poor substrates relative to glutamate. This Ser/Gly residue is located between the bound substrate and one of the cation binding sites, which provides an explanation for the coupling of substrate and cation binding.

Kinetic Rationale for Selectivity toward N- and C-terminal Oxygen-dependent Degradation Domain Substrates Mediated by a Loop Region of Hypoxia-Inducible Factor Prolyl Hydroxylases

Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the alpha-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two beta-strands (beta2 and beta3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the beta2beta3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the beta2beta3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.

Non-homologous Recombination of Deoxyribonucleoside Kinases from Human and Drosophila melanogaster Yields Human-like Enzymes with Novel Activities

In antiviral and cancer therapy, deoxyribonucleoside kinases (dNKs) are often the rate-limiting step in activating nucleoside analog (NA) prodrugs into their cytotoxic, phosphorylated forms. We have constructed libraries of hybrid enzymes by non-homologous recombination of the pyrimidine-specific human thymidine kinase 2 and the broad-specificity dNK from Drosophila melanogaster; their low sequence identity has precluded engineering by conventional, homology-dependent shuffling techniques. From these libraries, we identified chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2′,3′-didehydro-3′-deoxythymidine (d4T). These results demonstrate the potential of non-homologous recombination within the dNK family for creating enzymes with new and improved activities towards nucleoside analogs. In addition, our results exposed a previously unknown role for the C-terminal regions of these dNKs in determining substrate selectivity.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.

Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing

Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in all cells. AAA+ ATPases in these proteases bind protein substrates and power their mechanical denaturation and subsequent translocation into a secluded degradation chamber where polypeptide cleavage occurs. Here, we show that model unfolded substrates are engaged rapidly by ClpXP and are then spooled into the degradation chamber at a rate proportional to their length. Degradation and competition studies indicate that ClpXP initially binds native and unfolded substrates similarly. However, stable native substrates then partition between frequent release and infrequent denaturation, with only the latter step resulting in committed degradation. During degradation of a fusion protein with three tandem native domains, partially degraded species with one and two intact domains accumulated. These processed proteins were not bound to the enzyme, showing that release can occur even after translocation and degradation of a substrate have commenced. The release of stable substrates and committed engagement of denatured or unstable native molecules ensures that ClpXP degrades less stable substrates in a population preferentially. This mechanism prevents trapping of the enzyme in futile degradation attempts and ensures that the energy of ATP hydrolysis is used efficiently for protein degradation.

Crystal Structure of Mouse Carnitine Octanoyltransferase and Molecular Determinants of Substrate Selectivity*[boxs

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.

ASSESSMENT OF ARGININE 97 AND LYSINE 72 AS DETERMINANTS OF SUBSTRATE SPECIFICITY IN CYTOCHROME P450 2C9 (CYP2C9)

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wild-type and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.

Mouse Liver and Kidney Carboxylesterase (M-LK) Rapidly Hydrolyzes Antitumor Prodrug Irinotecan and the N-Terminal Three Quarter Sequence Determines Substrate Selectivity

Antitumor prodrug irinotecan is used for a variety of malignancies such as colorectal cancer. It is hydrolyzed to the metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which exerts its antineoplastic effect. Several human and rodent carboxylesterases are shown to hydrolyze irinotecan, but the overall activity varies from enzyme to enzyme. This report describes a novel mouse liver and kidney carboxylesterase (M-LK) that is highly active toward this prodrug. Northern analyses demonstrated that M-LK was abundantly expressed in the liver and kidney and slightly in the intestine and lung. Lysates from M-LK transfected cells exhibited a markedly higher activity on irinotecan hydrolysis than lysates from the cells transfected with mouse triacylglycerol hydrolase (TGH) (6.9 versus 1.3 pmol/mg/min). Based on the immunostaining intensity with purified rat hydrolase A, M-LK had a specific activity of 173 pmol/mg/min, which ranked it as one of the most efficient esterases known to hydrolyze irinotecan. A chimeric carboxylesterase and its wild-type enzyme (e.g., M-LKn and M-LK), sharing three quarters of the entire sequence from the N-terminus, exhibited the same substrate preference toward irinotecan and two other substrates, suggesting that the N-terminal sequence determines substrate selectivity. M-LK transfected cells manifested more severe cytotoxicity than TGH transfected cells upon being exposed to irinotecan. Topoisomerase I inhibitors such as irinotecan represent a promising class of anticancer drugs. Identification of M-LK as an efficient carboxylesterase to activate irinotecan provides additional sequence information to locate residues involved in irinotecan hydrolysis and thus facilitates the design of new analogs.

Determining substrate selectivity and regiochemistry of nitrilium phosphanylid tungsten complexes in cycloaddition reactions with terminal alkynes

Here we report reactions of intermediate nitrilium phosphanylid complexes, formed via reactions of a 2 H-azaphosphirene complex, and 2-furanocarbonitrile, 2-thiopheno-carbonitrile or 2-cyano-1,5-dimethyl-pyrrole, and different alkynes. These examples show the dependencies of substrate selectivity and regiochemistry of this new reactive intermediate. Furthermore, we report on an unusual 13C NMR coupling phenomena within the 2 H-1,2-azaphosphole ring.

Functional significance of N- and C-terminus of the amino acid transporters EAAC1 and ASCT1: characterization of chimeric transporters

To localize functionally significant domains in the amino acid transporters of mouse brain mEAAC1 and mASCT1, cRNA encoding for wild-type and chimeric transporters was injected into Xenopus oocytes. Activity of expressed transporters was investigated by measurements of uptake of 3H-labeled glutamate and serine and of glutamate- and serine-induced currents under voltage clamp. Though all transporters accept glutamate and serine as substrate, the central part of the protein (Ala 94-Met 418 of mEAAC1 and Ala 119-Ile 393 of mASCT1) determines substrate selectivity. The C-terminus rectifies the interaction with the respective substrate. A channel mode of the glutamate transporter can be activated by glutamate and serine, and the N- and C-termini of the mEAAC1 seem to be essential for the channel formation.

Structure-function analysis of liver-type (GLUT2) and brain-type (GLUT3) glucose transporters: expression of chimeric transporters in Xenopus oocytes suggests an important role for putative transmembrane helix 7 in determining substrate selectivity.

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (K(m) values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, GLUT3 is not) [Gould, G. W., Thomas, H. M., Jess, T. J., & Bell, G. I. (1991) Biochemistry 30, 5139-5145]. We have generated a range of chimeric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involved in substrate recognition and binding. The functional characteristics of these chimeras were determined by expression in Xenopus oocytes after microinjection of cRNA. Replacement of the region from the start of putative transmembrane helix 7 to the C-terminus of GLUT3 with the corresponding region from GLUT2 results in a chimera with the ability to transport fructose and exhibits a K(m) for 2-deoxyglucose transport of close to that observed for wild-type GLUT2 (8.3 +/- 0.3 mM compared to 11.2 +/- 1.1 mM). Replacement of the region in GLUT3 from the end of helix 7 to the C-terminus with the corresponding region from GLUT2 resulted in a species which was unable to transport fructose and whose K(m) for 2-deoxyglucose was indistinguishable from wild-type GLUT3. We have determined the affinity for 2-deoxyglucose, D-fructose, and D-galactose of these and other chimeras. In addition, the Ki for maltose, a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. The results obtained support a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported substrate and in which this region plays an important role in determining the K(m) for 2-deoxyglucose. Additional data is presented which suggests that a region between the end of putative transmembrane helix 7 and the end of helix 10, together with sequences in the N-terminal half of the protein may also participate in substrate recognition and transport catalysis.