Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Inhibition of brain energy metabolism by the alpha-keto acids accumulating in maple syrup urine disease

Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.     

Detection of heterozygotes in maple-syrup-urine disease: measurements of branched-chain alpha-ketoacid dehydrogenase and its components in cell cultures.

To detect heterozygotes for maple-syrup-urine disease (MSUD), activities of branched-chain-alpha-ketoacid (BCKA) dehydrogenase and its components in skin fibroblasts of two obligatory heterozygotes and amnion cells of a fetus at risk were measured. Intact heterozygous cells were found to decarboxylate [1-14C] alpha-ketoisovalerate at rates equal to or only slightly lower than normal subjects. The inability to differentiate heterozygotes from normals with the intact cell assay confirms earlier studies with intact leukocytes using [1-14C]leucine as substrate. By contrast, measurements of BCKA dehydrogenase activity with disrupted cell suspensions showed MSUD heterozygotes with 30%--60% of normal activity. Moreover, biphasic kinetics in heterozygous cells were observed with increasing substrate concentrations. The altered biphasic kinetics probably reflect expression of the normal allele in the early hyperbolic portion of the curve of the mutant allele in the later secondary rise at high substrate concentrations. Assays of component activities showed concordant E1 decarboxylase deficiency in both heterozygous- and homozygous-affected cells, whereas the E3, dihydrolipoyl dehydrogenase-component, activity was normal. The above results taken together appear to provide an approach to detection of the heterozygote in MSUD.     

Study on established lymphoid cells in maple syrup urine disease. Correlation with clinical heterogeneity

Summary Branched-chain keto acid dehydrogenase complex (BCKAD) was measured in lymphoid cells established from five patients with maple syrup urine disease (MSUD) and six control subjects. Two other MSUD lymphoid cell lines obtained from The Human Genetic Mutant Cell Repository were used as references. One day after subculture, the cells grew logarithmically up to 4–5 days. With this cell growth, BCKAD activity increased greatly in controls, but not in MSUD cells. The maximum BCKAD activity of MSUD cells was less than 7% and 13%–16% of the control in classic and variatant types, respectively. Leucine added to culture medium at the concentration of 10–20 mM significantly inhibited cell growth in MSUD cells alone, and with increasing concentration and impaired enzyme activity in a cell line, the effect became more prominent. The effects of isoleucine and valine were mild and did not differ between control and MSUD cells.This study was supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan (56480191; Matsuda)     

Variant maple syrupurine disease in mother and daughter

Intermittent MSUD in a mother and her daughter is reported. Fibroblast cultures were studied for branched-chain keto acid decarboxylase and results show that the mother has approximately 12% while the daughter has 5% of the normal enzyme activity. Other key members in the family were also studied for enzyme activity. It appears that the child has inherited an abnormal gene from her homozygous mother and another abnormal gene from her heterozygous father.A classification based on the degree of residual enzyme activity and protein tolerance places the mother in grade III and the daughter in grade II category. Classical MSUD, where the enzyme activity is less than 2% of normal, belongs to grade I.     

Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production

Kynurenine aminotransferase (KAT) catalyzes the formation of kynurenic acid (KYNA), the natural antagonist of ionotropic glutamate receptors. This study tests potential substrates and assesses the effects of amino acids and keto acids on the activity of mosquito KAT. Various keto acids, when simultaneously present in the same reaction mixture, display a combined effect on KAT catalyzed KYNA production. Moreover, methionine and glutamine show inhibitory effects on KAT activity, while cysteine functions as either an antagonist or an inhibitor depending on the concentration. Therefore, the overall level of keto acids and cysteine might modulate the KYNA synthesis. Results from this study will be useful in the study of KAT regulation in other animals.     

Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. Immunochemical analysis of the enzyme complex

Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.     

Occurrence of a Tyr393----Asn (Y393N) mutation in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex in maple syrup urine disease patients from a Mennonite population

Maple syrup urine disease (MSUD) is caused by a deficiency in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The incidence of MSUD in the Philadelphia Mennonites is 1/176 births resulting from consanguinity. In this study, we amplified cDNAs for the decarboxylase E1 alpha subunit of the branched-chain alpha-keto acid dehydrogenase complex from a classical MSUD patient and from an obligatory heterozygote of a Mennonite family by the PCR. Sequencing of the amplified cDNAs disclosed at codon 393 of the mature E1 alpha polypeptide a base substitution changing a tyrosine (encoded by TAC) to an asparagine residue (encoded by AAC), which is designated Y393N. A segment of the E1 alpha gene containing the 5' portion of exon 9 was amplified. Probing of the amplified genomic DNA with allele-specific oligonucleotide probes showed that the mutation in the E1 alpha gene was homozygous in six Mennonites affected with classical MSUD and was present in heterozygous carriers. The identification of the MSUD mutation in the Philadelphia Mennonites will facilitate diagnosis and carrier detection for this population.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Inactivation of the 2-ketoglutarate and pyruvate dehydrogenase complexes of beef heart by branched chain keto acids

Incubation of 2-ketoglutarate dehydrogenase complex with 2-ketoisovalerate, 2-keto-4-methylvalerate, or 2-keto-3-methylvalerate leads to the appearance of a lag phase and of a progressive loss of activity in subsequent measurements of the initial rate of oxidation of 2-ketoglutarate. In the case of 2-ketoisovalerate these effects are shown to be due to the formation of an isobutyryllipoate derivative of the enzyme, as a result of the very slow oxidation of 2-ketoisovalerate by the enzyme complex (Vmax congruent to 0.15% of that for 2-ketoglutarate). Incubation of the enzyme complex with 2-keto[14C]isovalerate or 2-keto[14C]glutarate results in comparable incorporation of radioactivity, amounting to 3.5 to 5.3 nmol of isobutyryl or succinyl residues per mg of protein in the complex. Isobutyryl residues are also incorporated in the enzyme during the simultaneous oxidation of both of these substrates. During the early phase of incubation of the complex with 2-ketoisovalerate the incorporation of isobutyryl residues is much faster than the loss of enzyme activity. This observation seems to support the suggestion that each 2-ketoglutarate decarboxylase subunit of the complex may catalyze the succinylation of more than one lipoate succinyltransferase subunit. Results are also presented showing the inactivation of pyruvate dehydrogenase complex on preincubation with 2-ketoisovalerate and of 2-ketoglutarate dehydrogenase complex with methylenecyclopropylpyruvate, the keto acid corresponding to the toxic amino acid hypoglycin. The relevance of covalent modifications of the two keto acid dehydrogenase complexes to the pathological manifestations of maple syrup urine disease are discussed.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

Mechanism of increased conversion of branched chain keto acid dehydrogenase from inactive to active form by a medium chain fatty acid (octanoate) in skeletal muscle.

We and others have previously shown that octanoate increases the oxidation of branched chain amino acids (BCAA) in skeletal muscle. The present study was designed to investigate the mechanism of this increased oxidation. Studies were performed with rat hind limbs perfused with 0.50 mM L-[1-14C]leucine with or without octanoate. The flux through branched chain keto acid (BCKA) dehydrogenase was measured, and the basal and total activity of BCKA dehydrogenase in skeletal muscle was determined. The rate of flux through BCKA dehydrogenase increased by 37, 119, and 297% with 0.5, 1.0, and 2.0 mM octanoate, respectively. This increase in flux was not due to a change in BCAA aminotransferase activity but was due to an increase in the basal activity of BCKA dehydrogenase. There was a strong correlation (r = 0.96) between increases in flux through BCKA dehydrogenase and increases in the basal activities of BCKA dehydrogenase. Preincubation of BCKA dehydrogenase with Mg2+ caused full activation of this enzyme, but preincubation with octanoate did not activate this enzyme. On the other hand, octanoate completely prevented the ATP-dependent inactivation of fully activated BCKA dehydrogenase. We conclude that octanoate increases the oxidation of leucine in skeletal muscle by increasing the activation of BCKA dehydrogenase. The mechanism of this activation is the inhibition of BCKA dehydrogenase kinase rather than the stimulation of a specific or nonspecific protein phosphatase.     

Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with L-[1-13C]phenylalanine

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defects in the mitochondrial multienzyme complex branched-chain alpha-keto acid dehydrogenase (BCKD; EC 1.2.4.4), responsible for the oxidative decarboxylation of the branched-chain ketoacids (BCKA) derived from the branched-chain amino acids (BCAA) leucine, valine, and isoleucine. Deficiency of the enzyme results in increased concentrations of the BCAA and BCKA in body cells and fluids. The treatment of the disease is aimed at keeping the concentration of BCAA below the toxic concentrations, primarily by dietary restriction of BCAA intake. The objective of this study was to determine the total BCAA requirements of patients with classical MSUD caused by marked deficiency of BCKD by use of the indicator amino acid oxidation (IAAO) technique. Five MSUD patients from the MSUD clinic of The Hospital for Sick Children participated in the study. Each was randomly assigned to different intakes of BCAA mixture (0, 20, 30, 50, 60, 70, 90, 110, and 130 mg.kg(-1).day(-1)), in which the relative proportion of BCAA was the same as that in egg protein. Total BCAA requirement was determined by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2). The mean total BCAA requirement was estimated using a two-phase linear regression crossover analysis, which showed that the mean total BCAA requirement was 45 mg.kg(-1).day(-1), with the safe level of intake (upper 95% confidence interval) at 62 mg.kg(-1).day(-1). This is the first time BCAA requirements in patients with MSUD have been determined directly.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

Inhibition of 3,4-dihydroxy-l-phenylalanine decarboxylase in rat striatal synaptosomes by amino acids interacting with substrate transport

Dopamine synthesis from 3,4-dihydroxy-l-phenylalanine in rat striatal synaptosomes was inhibited by a number of amino acids with aromatic or large aliphatic side chains. Inhibition was not seen when aromatic amino acid decarboxylase activity was measured in disrupted synaptosomes. Similarly, inhibition of dopamine synthesis from tyrosine was seen in the presence of leucine. The inhibition most likely results from interactions of the amino acids with substrate transport across the synaptosome plasma membrane, rather than directly with the catalytic enzymes. The kinetic data obtained are used to infer information about the relevant transport process; they suggest the potential importance of amino acid efflux as a regulatory step.     

Alternate mechanism for amino acid entry into Neurospora crassa: extracellular deamination and subsequent keto acid transport

The growth of the pm nbg mutant strain of Neurospora crassa was inhibited by the amino acid analog para-fluorophenylalanine despite the fact that none of the three constitutive amino acid permeases is functional in this strain. This observation led to the detection of both a deaminase which was released into the growth medium in response to para-fluorophenylalanine and a keto acid transport system which allowed entry of the resulting keto acid into the cell. The transported keto acid was recovered in cellular protein, suggesting its regeneration as the amino acid. The cooperative activity of these two systems represents an additional mechanism for the intracellular accumulation of amino acids, which is distinct from the known amino acid permeases.     

Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.     

From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.