Phenylalanine Hydroxylase from Chromobacterium violaceum PURIFICATION AND CHARACTERIZATION*

Phenylalanine hydroxylase was purified approximately 3000-fold to apparent homogeneity with a 13% yield and crystallized from L-phenylalanine-induced cells of Chromobacterium violaceum The enzyme was shown to be composed of a single polypeptide chain with an estimated molecular weight of approximately 32,000. Some of the physical properties of the enzyme include: a Stokes radius of 26.0 A, a sedimentation coefficient of 2.71 S, a diffusion coefficient of 8.20 x lo-’ cm’/s, a frictional ratio of 1.23, and an isoelectric point of pH 4.5. No detectable iron was found in the purified enzyme. Apparent K,,, values for L-phenylalanine and a-amino - 4 - hydroxy - 6,7 - dimethyltetrahydropteridine were 140 and 54 pM, respectively.


Phenylalanine
Hydroxylase from Chromobacterium violaceum PURIFICATION AND CHARACTERIZATION* (Received for publication, August 21, 1978) Hiroyasu Nakata, Takashi Yamauchi, and Hitoshi Fujisawa From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-11, Japan Phenylalanine hydroxylase was purified approximately 3000-fold to apparent homogeneity with a 13% yield and crystallized from L-phenylalanine-induced cells of Chromobacterium violaceum The enzyme was shown to be composed of a single polypeptide chain with an estimated molecular weight of approximately 32,000. Some of the physical properties of the enzyme include: a Stokes radius of 26.0 A, a sedimentation coefficient of 2.71 S, a diffusion coefficient of 8.20 x lo-' cm'/s, a frictional ratio of 1.23, and an isoelectric point of pH 4.5. No detectable iron was found in the purified enzyme. Apparent K,,, values for L-phenylalanine and a-amino -4 -hydroxy -6,7 -dimethyltetrahydropteridine were 140 and 54 pM, respectively.
Phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine: oxygen oxidoreductase (4 -hydroxylating), EC 1.14.16.1) is a monooxygenase which catalyzes the conversion of L-phenylalanine to L-tyrosine using tetrahydropterin as a reducing agent and molecular oxygen as an oxidizing agent. Tyrosine hydroxylase and tryptophan hydroxylase also require a reduced unconjugated pterin cofactor as an electron source and, in this regard, these three enzymes are believed to be similar in their mode of action (1). Although the most extensive studies on reaction mechanism and physical properties have been carried out on phenylalanine hydroxylase, the pure enzyme has not been available in large quantities for use in studying the reaction mechanism. Therefore, in order to study the reaction mechanism of pterin-requiring hydroxylase, it seems desirable to obtain the enzyme in pure form.
Recently, Guroff and his co-workers (2) reported that Chromobacterium violaceum contained phenylalanine hydroxylase and a cell-free phenylalanine hydroxylase was easily obtained from this organism. This paper describes a procedure for the complete purification of phenylalanine hydroxylase from C. violaceum. Some of its physical, chemical, and catalytic properties have been examined.

EXPERIMENTAL PROCEDURES
The "Experimental Procedures" and part of the "Results" are described in the miniprint supplement.' * This research was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' The "Experimental Procedures," part of the "Results," Figs. 1 to 5, Tables III and IV, and "References" are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from RESULTS Purity of Enzyme-A typical purification procedure of phenylalanine hydroxylase from C. violaceum was summarized in Table I. The yield of the enzyme increased almost 2fold after the step of DEAE-cellulose chromatography. By this procedure, the enzyme with a specific activity of 13.2 units/mg of protein was obtained in an overall purification of about 3000-fold with a 13% yield.
The purified enzyme gave a single protein band upon disc gel electrophoresis as shown in Fig. IA. A single band was also found in gel electrophoresis in 0.1% sodium dodecyl sulfate as shown in Fig. 1B.
The purified enzyme was crystallized by vapor diffusion technique using ammonium sulfate (22). The crystals took the form of rectangular plates as shown in Fig. 2. Physical Parameters-The molecular weight of the enzyme was determined by a number of procedures. The value of 32,400 was obtained from Ultrogel AcA 44 gel filtration, as shown in Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a molecular weight of 33,000 (Fig. 4), which was in good agreement with the value determined by gel filtration on Ultrogel AcA 44, indicating that phenylalanine hydroxylase from C. violaceum consisted of a single polypeptide chain. The results of sedimentation equilibrium studies were plotted as the logarithm of the equilibrium protein concentration (In A280 ",,,) versus the square of radial distance from the center of the rotor (?) as shown in Fig. 5. Taking a partial specific volume of 0.739 cm3/g, calculated as described below, together with the slope from Fig. 5, a molecular weight of 31,200 was obtained for the enzyme. The sedimentation velocity of the enzyme was measured by analytical centrifugation. A single, symmetrical schlieren peak was observed to sediment at spo,w = 2.71 S. Gel filtration on Ultrogel AcA 44 (Fig. 3) gave a Stokes radius of 26.0 A and the diffusion coefficient, &+,, was calculated to be 8.20 x 10m7 cm'/s from the value of the Stokes radius. A molecular weight of 31,700 was calculated from these values and a partial specific volume of 0.739 cm3/g was determined by the Svedberg equation. A frictional ratio of the enzyme was calculated to be 1.23, indicating that the enzyme was nearly spherical in shape. The physical parameters of phenylalanine hydroxylase from C. violaceum are summarized in Table II. Polyacrylamide gel electrofocusing showed a single protein band and the isoelectric point which was estimated to be 4.5.
The absorption spectrum of the enzyme represented a typical absorption pattern of protein solution, with an absorption maximum at 279 nm and a small shoulder at 290 nm. The enzyme exhibited no absorption in the visible range.    Table III. Since 3 mol of half-cystine/mol of enzyme were determined as cysteic acid after performic acid oxidation and three sulfhydryl groups/m01 of enzyme were determined by the use of 5,5'-dithiobis(2-nitrobenzoic acid) in 2% sodium dodecyl sulfate, there appeared to be no disulfide linkage in the enzyme. When the native enzyme was titrated with 5,5'dithiobis(2-nitrobenzoic acid), only 1 mol of sulfhydryl residue/mol of enzyme reacted with the reagent. Therefore, it appeared that 2 mol of cysteine were buried inside the enzyme molecule. A partial specific volume of 0.739 cm3/g was estimated from the amino acid composition (23). The iron content of the enzyme was determined by the atomic absorption spectrophotometer.
Prior to determination, the purified enzyme (0.37 mg/ml) was dialyzed overnight at 4°C against 50 mM 2-(N-morpholino)ethanesulfonic acid/ NaOH buffer, pH 6.5, without an appreciable loss of the activity. No detectable iron was found in this preparation.

Catalytic
Properties-The optimum pH for the enzyme activity was found to be in the range of 7.3 to 7.5. The apparent & values for L-phenylalanine and DMPH4' were estimated from the linear double reciprocal plots to be ap-' The abbreviations used are: DMPH,, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine; GMPH,, 2-amino+hydroxy-6-methyltetrahydropteridine.
proximately 140 and 54 PM, respectively. The apparent V,,, value was also estimated to be approximately 14 pmol of tyrosine/min/mg of protein from the double reciprocal plots. Under the conditions described under "Experimental Procedures," the rate of hydroxylation of L-tryptophan was found to be approximately 0.4% of that of L-phenylalanine. No detectable activity of tyrosine hydroxylation was observed. DMPH4, 6MPHI, and tetrahydrofolate served as electron donors for the enzyme. DMPH, was found to be the most active among these three synthetic pteridines.
The enzyme activity with GMPH, and tetrahydrofolate was approximately 60 and 10% of that with DMPHI, respectively.
Stoichiometry of the enzyme reaction is presented in Table  IV. L-Phenylalanine was converted to a stoichiometric amount of L-tyrosine with the consumption of equimolar amounts of oxygen and DMPH,.

DISCUSSION
In the present study, phenylalanine hydroxylase was obtained for the first time as a homogeneous and crystalline preparation.
Since Letendre et al.
(2) reported that C. violaceum contained a phenylalanine hydroxylase and a cell-free preparation was easily obtained from this organism, it was anticipated that the bacterium contained a relatively large amount of phenylalanine hydroxylase. However, a 3000-fold purification was required for obtaining a homogeneous enzyme preparation.
The molecular weight of the enzyme was determined by a number of procedures as summarized in Table II. The values ranging from 31,200 to 32,400 were consistent with the value of 33,000 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that the enzyme consisted of a single polypeptide chain. Letendre et al. (24) reported that phenylalanine hydroxylase from Pseudomonas species was a single protein chain of molecular weight between 25,000 and 27,000. In contrast to these bacterial enzymes, phenylalanine hydroxylases from mammalian livers are believed to exist in polymeric forms (4, 25, 26). Rat liver enzyme consisted of a dimer or tetramer of subunit of J4, = 55,000 (25), human liver enzyme consisted of a dimer of subunit of M, = 54,000 (4), and monkey liver enzyme consisted of two proteins of very similar molecular weight between 45,000 and 57,500 (26).
Hydroxylation of tryptophan by rat liver phenylalanine hydroxylase was demonstrated by Renson et al. (27) using partially purified enzyme and confirmed by Kaufman and Fisher (1) using highly purified (90% pure) enzyme. Bacterial phenylalanine hydroxylase from C. violaceum also catalyzed hydroxylation of L-tryptophan, although it was much slower than that of L-phenylalanine.
Kaufman and his co-workers (28) demonstrated that phenylalanine hydroxylase of rat liver was an iron enzyme and the metal might be involved in the enzymic reaction. Bacterial phenylalanine hydroxylase from Pseudomonas species was shown to be activated by mercuric, cadmium, cupric, and cuprous ions as well as ferrous ions (29). Contrary to our expectations, no iron was detected in phenylalanine hydroxylase from C. violaceum by atomic absorption spectroscopy. Further investigations on the involvement of iron or other metals in the enzymic reaction are now in progress.