The isolation and properties of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.     

Crystallization and preliminary X-ray analysis of the methane monooxygenase hydroxylase protein from Methylococcus capsulatus (Bath).

Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition alpha 2 beta 2 gamma 2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2(1)2(1)2(1), with one dimer of relative molecular mass M(r) = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 A, b = 110.1 A, c = 333.5 A. The crystals diffract uniformly beyond 2.5 A resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.     

Crystallization and preliminary X-ray analysis of porin from Rhodobacter capsulatus

Porin monomers of the phototrophic bacterium Rhodobacter capsulatus were purified. Crystals were obtained from a solution of porin solubilized with the detergent octyltetraoxyethylene within 5 days using the vapor phase equilibration technique. The crystals were rhombohedral with an edge length of 0.4 mm. The space group was trigonal R3 with unit cell constants of a = b = 95 A, c = 147 A. Reflexions were observed to 3.2 A.     

Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver

1. Pteridine cofactor of phenylalanine hydroxylase (EC 1.14.16.1) and dihydropteridine reductase (EC 1.6.99.7) in the phenylalanine hydroxylating system have been studied in the fetal rat liver. 2. Activities of pteridine cofactor and dihydropteridine reductase were measured as about 6 and 50%, respectively, of the levels of adult liver in the liver from fetuses on 20 days of gestation, at this stage the activity of phenylalanine hydroxylase was almost negligible in the liver. 3. Development of the activity of sepiapterin reductase (EC 1.1.1.153), an enzyme involved in the biosynthesis of pteridine cofactor, was studied in rat liver during fetal (20-22 days of gestation), neonatal and adult stages comparing with the activity of dihydrofolate reductase (EC 1.5.1.3). Activities of the enzymes were about 80 and 50%, respectively, of the adult levels at 20 days of gestation. 4. Some characteristics of sepiapterin reductase and dihydropteridine reductase of fetal liver were reported.     

Hydroxylation of 4-methylphenylalanine by rat liver phenylalanine hydroxylase

Rat liver phenylalanine hydroxylase that has been activated with lysolecithin catalyzes the hydroxylation of 4-methylphenylalanine in the presence of a pterin cofactor. Two products, 4-hydroxymethylphenylalanine and 3-methyltyrosine, can be detected. The total amount of amino acids hydroxylated is equal to the amount of tetrahydropterin oxidized. Isotopic labeling studies with 18O2 and H2(18)O show that the hydroxyl groups of both products are derived from molecular oxygen and not from water. Results obtained with 2H-labeled substrates support the conclusion that these products are formed via different mechanistic pathways. Our previous investigations on substrate analogs, as well as the present results, indicate that a highly reactive oxygen-containing intermediate, such as an enzyme-bound iron-oxo compound, must be the hydroxylating species. Our present results could stimulate further discussion of the possibility that the reaction mechanism for the "NIH-shift" of the methyl group may not involve the spontaneous opening of an epoxide intermediate.     

Purification of rat liver phenylalanine hydroxylase by affinity chromatography

Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 m KCl in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases

Abstract

Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.     

Sequence comparison of rat liver phenylalanine hydroxylase and its cDNA clones

Classical phenylketonuria, an inborn error in metabolism, is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. The identification of putative cDNA clones coding for rat liver phenylalanine hydroxylase by hybrid-selected translation has previously been reported [Robson, K. J., Chandra, T., MacGillivray, R. T. A., & Woo, S. L. C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4701-4705]. The authenticity of the clones, however, could not be definitively ascertained at the time because of a lack of amino acid sequence data of the enzyme in the literature. Purified rat liver phenylalanine hydroxylase was subjected to cyanogen bromide treatment, and the resulting fragments were used for N-terminal amino acid sequence analysis. The partial amino acid sequence was then compared to that deduced from an open reading frame in the nucleotide sequence of the cDNA clones. A perfect match of 17 amino acid residues was found between the two sequences following a unique methionine codon present in the nucleotide sequence, thereby providing unambiguous evidence for the identity of the rat liver phenylalanine hydroxylase cDNA clones.     

Studies on the primary structure of rat liver phenylalanine hydroxylase

The primary structure of phenylalanine hydroxylase purified from rat liver was investigated with high speed gel filtration chromatography, cyanogen bromide cleavage and end group analyses of polypeptides derived from the enzyme. On gel filtration in the presence of 6M guanidine hydrochloride, the enzyme gave a single peak corresponding to a molecular weight of 52,000. In the same system the enzyme that had been cleaved with cyanogen bromide gave two peptides (CB1, Mr = 32,800 and CB2, Mr = 20,400). Sequence studies showed that the alignment of these two peptides was CB1 - CB2. Furthermore, in experiments using 32P phosphorylated enzyme, the site of phosphorylation by cAMP-dependent protein kinase was found to be located on the CB1 peptide. The NH2-terminus of this enzyme, which was found to be blocked, was shown to be N-acetylalanine. By both carboxypeptidase A digestion and hydrazinolysis, the carboxyl terminus was identified as serine. These data indicate that the phenylalanine hydroxylase molecule from rat liver is composed of subunits which are homogenous or, at least, very similar in their primary structure.     

Crystallization and a preliminary X-ray diffraction study of isozyme 3-3 of glutathione S-transferase from rat liver

Crystals of the homodimeric isozyme 3-3 of glutathione S-transferase from rat liver have been obtained with the hanging drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of the enzyme required the presence of both the enzyme inhibitor (9R, 10R)-9, 10-dihydro-9-(S-glutathionyl)-10-hydroxyphenanthrene and the detergent beta-octylglucopyranoside. The crystals belong to the monoclinic space group C2, with cell dimensions of a = 88.24(8) A, b = 69.44(4) A, c = 81.28(5) A, beta = 106.01(6) degrees, and contain four dimeric enzyme molecules per unit cell. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination at high resolution.     

Crystallization and preliminary X-ray analysis of aldehyde dehydrogenase from Vibrio harveyi.

Aldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+. The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three-dimensional structure that may be correlated to cofactor specificity. Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique. In order to improve crystal size and quality, iterative seeding techniques were employed. The crystals belong to space group P2(1), with unit cell dimensions a = 79.4 A, b = 131.1 A, c = 92.2 A, and beta = 92.4 degrees. Freezing the crystal to 100 K has enabled a complete set of data to be collected using a rotating anode source (lambda = 1.5418 A). The crystals diffract to a minimum d-spacing of 2.6 A resolution. Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit. Self-rotation functions show the presence of 3 noncrystallographic twofold symmetry axes.     

Crystallization and preliminary X-ray crystallographic analysis of lipase from Pseudomonas cepacia.

Large crystals of lipase from Pseudomonas cepacia have been grown at room temperature from solutions containing 2-methyl-2,4-pentanediol and sodium citrate. They grow within two weeks to typical dimensions of 1.0 mm x 0.5 mm x 0.3 mm. The crystals belong to the monoclinic space group P2(1), with unit cell parameters a = 84.91 A, b = 47.33 A, c = 86.00 A, and beta = 116.09 degrees. And they diffract to about 1.6 A upon exposure to synchroton X-rays. X-ray data have been collected to 2.2 A Bragg spacing from a native crystal.