Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104)

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.     

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells : III. Subcellular Distribution of Prolyl Hydroxylase

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(l-proline) in the presence of oxygen, α-ketoglutarate, ferrous ion, and ascorbate. The K_m for poly(l-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocollagen analog (Pro-Pro-Gly)₅·4H₂O.     

Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana

The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD⁺ oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K_m values for α-ketoglutarate, NADH, NH₄⁺, NAD⁺, and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the K_m for α-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K_m values for NADH and NAD⁺ were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between α-ketoglutarate and an ε-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH₄⁺-inducible glutamate dehydrogenase in this organism.     

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.     

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly⁵⁶, Thr⁵⁷), TMS3 (Glu¹³⁸), and TMS6 (Phe²⁴⁸), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent K_m for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties

Purified glutamine synthetase from pea seedlings was most active with Mg²⁺ as the metal activator, but Mn²⁺ and Co²⁺ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg²⁺ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co²⁺ also gave a sigmoidal saturation curve, but when Mn²⁺ was varied only slightly sigmoidal kinetics were seen. Addition of Mn²⁺, Ca²⁺, or Zn²⁺ at low concentrations sharply inhibited the Mg²⁺ -dependent activity, partially by shifting the pH optimum. Addition of Co²⁺ did not inhibit Mg²⁺-dependent activity. The nucleotide triphosphate specificity changed markedly when Co²⁺ or Mn²⁺ replaced Mg²⁺. Using the Mg²⁺-dependent assay, the Michaelis constant (Km) for NH₄⁺ was about 1.9 × 10⁻³ M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.     

Effect of ammonium and other ions on the glucose-dependent active transport of l-histidine in slices of rat-brain cortex

1. The influence of cations on the active transport into cells of rat-brain-cortex slices of l-histidine, an amino acid that is not metabolized by this tissue, has been studied. 2. Like other amino acids, l-histidine accumulated in the cells in the presence of glucose in concentrations up to over double that in the incubation medium. 3. The active transport of l-histidine was highest in a medium containing Ca²⁺ (3mm). The addition of K⁺ (27mm) led to a marked decrease in the intracellular concentration of l-histidine, though the oxygen uptake of the slices was higher. 4. The active l-histidine transport was inhibited by NH₄⁺. The inhibitory effect increased with the NH₄⁺ concentration, being about 25% at 8mm, 65% at 20mm, and 90% at 27 and 50mm. The oxygen uptake of the brain slices was depressed by only 25% by the highest NH₄⁺ concentration used, and less by lower concentrations.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

Inhibition of glucuronokinase by substrate analogs

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a K_i value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl α-glucuronoside, methyl β-glucuronoside, β-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The β-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.     

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.     

Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50–80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, K_m(ATP) 0.60mm, K_m(glucose) 0.042mm; peak II, K_m(ATP) 0.66mm, K_m(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, K_m(ATP) 0.56mm, K_m(glucose) 0.048mm; peak II, K_m(ATP) 0.68mm, K_m(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Nucleoside Diphosphate-sugar 4-Epimerases I. Uridine Diphosphate Glucose 4-Epimerase of Wheat Germ

Uridine diphosphate (UDP)-glucose 4-epimerase (EC 5.1.3.2) has been purified over 1000-fold from extracts of wheat germ by MnCl₂ treatment, (NH₄)₂SO₄ fractionation, Sephadex column chromatography, and adsorption onto and elution from calcium phosphate gel. The enzyme has a pH optimum of 9.0. Km values are 0.1 mm for UDP-d-galactose and 0.2 mm for UDP-d-glucose. NAD is required for activity; K_a = 0.04 mm. NADH is an inhibitor strictly competitive with NAD; K_i = 2 μm. Wheat germ also contains UDP-l-arabinose 4-epimerase (EC 5.1.3.5) and thymidine diphosphate (TDP)-glucose 4-epimerase which are distinct from UDP-glucose 4-epimerase.     

Regulation of sulfate transport in filamentous fungi

Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10⁻⁵ to 10⁻⁴m and a V_max (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the V_max value with no marked change in the Km. Azide and dinitrophenol also reduce the V_max.     

Serine Transhydroxymethylase of Cauliflower (Brassica oleracea var. botrytis L.): Partial Purification and Properties

Serine transhydroxymethylase (EC 2.1.2.1) has been purified 46-fold from cauliflower (Brassica oleracea var. botrytis L.). The enzyme was completely dependent on the presence of tetrahydrofolic acid for the conversion of serine to glycine. The addition of pyridoxal phosphate gave a large increase in the reaction rate. A double pH optimum was observed with maxima at 7.5 and 9.5. The enzyme is specific for l-serine. The d-isomer is neither a substrate nor an inhibitor. The Michaelis constants for l-serine, tetrahydrofolic acid, and pyridoxal phosphate were 300 μm, 760 μm, and 24 μm, respectively. The addition of K⁺ also stimulated the reaction rate considerably. The effect was quite specific since all other metal ions tested either had very little: influence or were extremely inhibitory.     

l-Phenylalanine Ammonia-Lyase (Maize): Evidence for a Common Catalytic Site for l-Phenylalanine and l-Tyrosine

l-Phenylalanine ammonia-lyase (E.C. 4.3.1.5) from maize is active with l-tyrosine and l-phenylalanine and exhibits atypical Michaelis-Menten kinetics with both substrates. With phenylalanine as a substrate, the pH optimum is 8.7 and with tyrosine, 7.7. The estimated Km at high substrate concentrations is 0.27 mm for phenylalanine and 0.029 mm for tyrosine. However, the V_max with phenylalanine is eight times higher than the V_max with tyrosine when both are measured at pH 8.7, and 7 times higher when both are measured at their pH optima. The following evidence leads us to the conclusion that there is a common catalytic site for both substrates: (a) It is impossible to appreciably alter the ratio of the two activities during purification and isoelectric focusing. (b) The ratio of the products formed in mixed substrate experiments is in good agreement with the ratio predicted from the estimated Km values. (c) NaBH₄ reduces both activities to the same degree and l-phenylalanine, l-tyrosine, cinnamate, and p-coumarate protect both activities against NaBH₄ reduction to the same degree. In contrast, the enzyme isolated from potato, which does not act on l-tyrosine, is not protected against reduction by either l-tyrosine or p-coumarate. However, both enzymes appear to have a dehydroalanine-containing prosthetic group.