Eur. J. Biochem. 210, 561 - 568 (1992) FEBS 1992

Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli Gabriele SCHOEDON1, Udo REDWEIK’, Gerhard FRANK’, Richard G . 11. COTTON3 and Neriad BLAIJ’

’

Division of Clinical Chemistry, Department of Pediatrics, University of Zurich, Switzerland Institute of Molecular Biophysics, Federal Institule of Technology, ETH Honggerbcrg, Zurich, Switzcrland The Murdoch Institutc, Royal Children’s Hospital, Melbourne, Australia

(Received July 7, 1992) - EJB 92 0965

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichiu colito absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase 1 has a relatively high apparent K,. The values are slightly different for enzyme purified by GTP-agarose (100 pM) and UTP-agarose (110 pM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in V,,, by K , divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+,had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of C a 2 + .In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 pM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobioptcrin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.

CTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. This is the first step in the multienzyme biosynthesis of the cofactor, tetrahydrobiopterin, of the aromatic amino acid hydroxylases [l]. GTP cyclohydrolase I has been described in many biological systems (for review see [2]). It has been purified to apparent homogeneity from Escherichiu coli [3], Drosophilu [4], and recently from human and rat liver [5, 61. The sequence of the gene coding for CTP cycylohydrolase I of E. coli and OK the adjacent region has been determined [I. The position of GTP cyclohydrolase I at the beginning of a multienzyme rcaction sequence makes it a logical candidate for regulation of tetrahydrobiopterin biosynthesis. Inhibition of GTP-cyclohydrolase-I activity by the metabolic end product, tetrahydrobiopterin, has been reported to take place in vim and in vivo [S, 91. Hormonal regulation of GTP cyclohydrolase 1 has been dcscribed for certain tissues [lo], and Currespondenre to N. Blau, Abteilung fur Klinische Chemie, Universit~ls-Kindcrklinik, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland Fax: +4112667171

Enzyme. GTP cyclohydrolase I (EC 3.5.4.16). Note. The novel amino acid sequence data published here have been submitted to the SWISSPROT sequence data bank(s).

regulation via substrate pool, e. g, intracellular GTP concentration, has been suggested in the cellular immune system [9]. In Drosophilu, the structural gene for GTP cyclohydrolase I has been cloned and the gene product was found to be essential for vital functions in embryogenesis and for eye pigmentation [Ill. However, the kinetics of GTP-cyclohydrolase enzyme activity have so far mostly been described as simple MichaelisMenten relationships [ 2 , 31, although factors altering the enzymic activity were observed 14, 61. GTP cyclohydrolase I from E. coli is commonly used for the production of dihydroneopterin triphosphate, the key substrate for enzymic studies of the further tetrahydrobiopterin biosynthetic steps. We prepared E. coli GTP cyclohydrolase I of the highest possible purity and studied this stable and highly active enzyme at the molecular and enzymological level. By direct measurement of the product, dihydroneopterin triphosphate, the kinetic characteristics of GTP-cyclohydrolase-I activity were investigated in the presence of various effector molecules. Using monoclonal antibodies of defined specificity, separate binding sites for GTP and effector molecules were demonstrated on the enzyme subunits by Western-blot analysis. The data presented in this study provide evidence for GTP cyclohydrolase I being an M-type allosteric regulatory en-

562 zyme. It might be a model for the regulation of GTP cyclohydrolase I and for the control of the tetrahydrobiopterin biosynthesis in mammals. MATERIALS AND METHODS Chemicals

GTP lithium salt, and UTP-agarose were from Sigma Chemical Co. ; dihydroneopterin triphosphate standard was obtained enzymically using GTP cyclohydrolase I immobilized on Tresyl-Sepharose according to [12]; neopterin and tetrahydrobiopterin were from Dr. B. Schircks Laboratories (Jona, Switzerland); GTP-agarose, DEAE A-50 anion-exchange resin and Ultrogel AcA 22 were from Pharmacia LKB Biotechnology ; Partisil-10 SAX anion-exchange column was from Whatman, Chemical Separation Division. E. coli strain B, MI135 lj2 log growth phase, was from Merck AG. All other chemicals used for buffcr solutions were from Merck AG . Purification of GTP cyclohydrolase 1 from E. coli

GTP cyclohydrolase I was purified from 500g E. coli with the following modifications to the previously described method [3]. First, a gel-filtration step was performed on Ultrogel AcA 22 prior to affinity chromatography on GTPagarosc. Secondly, aftinity chromatography was also performed on UTP-agarose [ 131. Briefly, the active pool from the DEAE A-50 ion-exchange column was concentrated in an Amicon ultrafiltration cell and applied t o a column of Ultrogel AcA 22 equilibrated with 50 mM potassium phosphate, pH 7.8. The column was eluted with the same buffer at a flow rate of 30ml/h and 10-ml fractions were collected. The fractions containing enzyme activity were concentrated to a volume of 130 ml. The Ultrogel pool was divided for affinity chromatography on GTPagarose or UTP-agarose. 50% of the volume of the Ultrogel pool was diluted tenfold with 10 mM potassium phosphate, pH 7.0 (buffer A), and applied to a column of UTP-agarose (1.6 cm x 2.6 cm) equilibrated with the same buffer. The column was washed with 300 ml buffer A containing 0.3 M KCl (buffer B) at a flow rate of 30 ml/h. The enzyme was eluted with 100 ml buffer B containing 50 mg UTP at a flow ratc of 10 ml/h. The active fractions were combined and concentrated to a volume of 3 ml. UTP was removed by chromatography on Sephadex G-25 medium. The enzyme preparations were analyzed by SDSjPAGE according to Laemmli [I 41. N-terminal sequence analysis

Amino-acid-sequence determination of E. coli GTP cyclohydrolase I was performed on a Knauer model 810 protein sequencer (Dr. Herbert Knauer GmbH), which was modified to enable isocratic identification of phenylthiohydantoinXaa after a previously described method [15]. Either the enzyme prcparations after affinity chromatography by GTPagarose or UTP-agarose, or the 25.5-kDa peptide isolated after SDSjPAGE and blotting on poly(viny1idene difluoride) membrane (Millipore Corp.) were used for sequence analysis. Internal sequences were obtained after in-situ cleavage with CNBr ofthe blotted 25.5-kDa subunit and subsequent separation of the resulting smaller peptides by SDSjPAGE and reblotting on poly(viny1idene difluoride) membrane.

Assay of GTP-cyclohydrolase-I activity

For basic kinetic studies, the standard reaction mixture contained 25 mM Tris/HCl, pH 8.0, enzyme (< 200 nM) and 0.05 - 2.0 mM GTP in a final volume of40 pl. The incubation was performed in the dark at 37 "C for 30 min and the reaction was terminated by addition of 20 p1 iodine solution (0.2% 12, 0.4% KI, in 20 mM HCl). After 1 min, 20 p1 0.5% ascorbic acid was added and the sample was deep frozen on dry ice. For HPLC analysis, 20 p1 sample was applied to a Partisil-10 SAX column (120 mm x 6 mm). Neopterin triphosphate was eluted with a solvent of 250 mM NH4H2P04, 250 mM KC1, and 50 mM Na4P207, pH 5.9. at a flow rate of 1 ml/min, and the products were detected fluorometrically (excitation 350 nm ; emission 450 nm). To study the influence of effectors on GTP-cyclohydrolase-I activity, the following assay was used. The reaction mixture contained 50 mM TrisjHCl or 50 mM potassium phosphate, pH 7.6, 0.1 M KC1, 0.2 mM GTP, effector and enzyme in a final volume of 100 p1. After incubation for S 30 rnin at room temperature with the effector, the reaction was performed at 37 "C for 45 min in the dark. The reaction was then stopped by addition of 100 p1 0.5 M NH4H2P04 and the samples were at once frozen on dry icejethanol. For HPLC analysis, 50 - 100 pl sample was injected immediately after thawing on a Partisil-10 SAX column (120 min x 6 mm) and eluted with 0.5 M NH4H2P04 and 0.5 M KC1, pH 4.0, at a flow rate of 1.0 ml/min. Dihydroneopterin triphosphate and GTP were detected simultaneously by ultraviolet light at 280 nm. 1 U GTP cyclohydrolase I is defined as the enzyme activity catalyzing the formation or 1 pmol dihydroneopterin triphosphate/min at 37°C. Western-blot analysis

Western-blot analysis of purificd GTP cyclohydrolase I was performed according to [16], using two monoclonal antibodies; the monoclonal antibody 1F11, which is related to the substrate binding site [S],and the monoclonal anti-idiotypic antibody NS7, mimicking the pterin cofactor [17]. For competition studies, the blots were incubated with GTP, UTP, or tetrahydrobiopterin. In brief, after blocking with 2% bovine serum albumin in Tris-buffered saline (buffer C), the nitrocellulose strips were incubated with 1 mM solutions of GTP, UTP, or tetrahydrobiopterin in 10 mM buffer C, pH 7.2, for 30 min at room temperature. The monoclonal antibodies were added in appropriate dilutions (NS7,l: 5000; 1F11, 1 : 100) in buffer C, and the blots were further incubated at 4°C overnight. After washing with buffer C/0.03% Tween-20 three times (10 min/wash), binding of monoclonal antibodies was detected by incubation with goat anti(mouse IgG/IgM) horse-radish-peroxidase conjugate diluted 1 : 3000 in buffer C/ 2% bovine serum albumin, for 1 h at room temperature. After washing with buffer C/0.03% Tween-20 three times (10 min/ wash), blots were developed with 4-chloro-1-naphthol for 15 min at room temperaturc.

RESULTS Purification of GTP cyclohydrolase I

The activity peak of the native enzyme complex is located at the position of 200 kDa in Ultroge! AcA 22 gel-filtration

563

kDa

97

66 43

A P r o - S e r - Leu - S e r - Lys -G1u -A1 a -A1 a - Leu-Val -

10

H i s - G1 u -A1 a - Leu-Val -A1 a - A r g -G1 y - Leu- G1 u -

20

Thr-Pro-Leu-Arg-Pro-Pro-Val-His-Glu-Met-

30

A s p - A s n - G1 u - T h r - A r g -Lys - S e r -Leu- I1 e - A1 a -

40

Gly-His-Met-Thr-Glu-Glu-Met-Gln-Leu-Leu-

50

B

31

22

14

Fig. 1. SDS/PACE analysis of CTP cyclohydrolase I. Fractions of different purification steps werc separated on a 12.5% polyacrylamide gel. (A), Molecular mass markers; (I), crudc cxtract; (2),ammoniumsulfate precipitate; (3), heat denaturation; (4), Sephadex DEAE-A50 pool; ( 5 ) , enzyme preparation after IJltrogel AcA 22; (6), enzyme preparation after UTP-agarose; (7), enzyme preparation after GTPagarose. Molecular masses of markers are indicated in kDa, on the right.

chromatography. Both the GTP-cyclohydrolase-I preparations after GTP-affinity or UTP-affinity chromatography showed a single peptide band of 25.5 kDa after SDSjPAGE on a 12.5% polyacrylamide gel (Fig. 1). The specific activities were 55 mU/mg protein for the GTP-affinity-purified enzyme and 102 mU/mg protein for the UTP-affinity-purified enzyme. When the enzyme activity was determined by the formic-acid-release assay, the specific activities for the two preparations were 3300 U/mg and 6100 U,’mg, respectively, compared with 700 U/mg reported in prior studies [3].

Val-Thr-Val-Arg-Asp-Ile-Thr-Leu-Thr-Ser-

10

Thr-Xaa-Glu-His-His-Phe-Val-Thr-Ile-Asp-

20

G1 y - Lys - A1 a - T h r - Va 1 - A1 a -Tyr - Xa a - Pro - Lys -

30

Fig. 2. Amino acid scquence of GTP cyclohydrolase 1. (A) Partial Nterminal amino acid sequence of GTP cyclohydrolase I from E. coli.. (b) Internal amino acid sequence of the GTP-cyclohydroalse-I subunit obtained by CNBr cleavage.

Table 1. Western-blot analysis of GTP cyclohydrolase I purified on UTP-agarose or GTP-agarose. Monoclonal antibodies with different specificities, e. g. NS7 specific to the tetrahydrobiopterin-binding site, and 1F11, related to the substrate-binding site, were used. GTP, UTP and tetrahydrobiopterin were incubated together with the antibodies at a concentration of 1 mM each at 4°C overnight; prior incubation time with 1 mM effector solution was 30 min. BH4, tetrahydrobioptcrin. Antibody/incubation

IF11 lFll/UTP 1F11/GTP IFll/BH4 NS7 NS7IUTP NS7/GTP NS7/BH4

Enzyme purified with UTP-agarose

GTP-agarosc

+++ +++ + +++

+++ +++ + +++ +++ +++ +

-

-

-

N-terminal sequence analysis

The N-terminal sequence of more than 50 residues was obtained by automated Edman degradation of both the native GTP-affinity-purified and UTP-affinity-purified enzyme. When the 25.5-kDa peptide band of GTP cyclohydrolase I was blotted onto poly(viny1idene difluoride) membrane (20 pmol/ band) and subsequently sequenced, the same N-terminal sequence was obtained. The first 50 residues are shown in Fig. 2a. The sequence data show that the enzyme preparation is essentially homogeneous and that the enzyme complex is composed of identical subunits. There is also agreement with the earlier finding of proline being the N-terminal amino acid

PI.

Internal sequence information was obtained by CNBr cleavage of the GTP-cyclohydrolase-I subunit. The predominant peptide had a molecular mass of approximately 14 kDa and yielded a sequence of 30 residues from poly(viny1idene difluoride) blot, shown in Fig. 2 b.

Western-blot analysis

When affinity-purified GTP cyclohydrolase I was analyzed by Western blotting using the monoclonal antibodies 1F11 (related to the GTP-binding site) and NS7 (related to the tetrahydrobiopterin-binding site), the preparation purified by using the UTP affinity column did not react with the NS7 antibody. The preparation from the GTP affinity column reacted well with antibody NS7. Both the preparations from GTP and UTP affinity reacted with the 1F11 antibody. The results obtained by Western-blot analysis are summarized in Table 1. Incubation of the GTP-purified preparation with NS7 in the presence of UTP abolished rcactivity with the NS7 antibody. In the presence of tetrahydrobiopterin, NS7 showed only weak reactivity. The same was true for antibody 1F11 in the presence of GTP.

564

a 8 U

2 loo--

5g

v

100

0

n I-

: U

50 I

100

500

1000

1500

2000

n

2500

?4

I

z

10

b

Time, rnin

I

I

100

50

10

IF /

without KCI

1

Fig. 4. The effect of the divalent cation, M g 2 + ,on the enzymic reaction of GTP cyclohydrolase I. The reaction was carried out as described in the Materials and Methods for effector studies. The incubation buffer contained 0.1 M KCl and UTP-agarose-purified cnzymc. The reaction was stopped at the times indicated and the samples deep frozen. The concentration of dihydroneopterin triphosphate (NH,TP) formed and GTP consumed were measured simultaneously by HPLC and ultraviolet detection at 280 nm. ( O ) ,without M g 2 + ;( O ) ,with 8 m M Mg2+.

insensitive to changes of GTP concentration (Fig. 3 a, b). No positive cooperation was observed. The apparent K,,, is very similar for both preparations: 100 pM for the GTP-affinity-purified preparation and 110 pM for the UTP-affinity-purified preparation. The saturation properties were different for the two preparations: GTP cyclohydrolase I after GTP affinity required a 160-fold increase in substrate concentration to reach 90% maximal velocity, while GTP cyclohydrolase I after UTP affinity required only a 75-fold higher substrate concentration (Fig. 3 a, b). As shown in Fig. 3c, incubation of UTP-purified cyclohydrolase in the presence of KCl in the reaction buffer produced an increase in V,,,,,, but no change in apparent K,. The KCl concentration yielding maximal activity was 100 mM in 50 mM Tris/HCl or phosphate buffers. KC1 had the same effect on the GTP-purified enzyme (data not shown). The molecular activity of the UTP-affinity-purificd GTP cyclohydrolase I was calculated to be 21/inin, as V,,, was 102 nmol . min . mg protein and the molecular mass 210 kDa. The molecular activity of the GTP-affinity-purified GTP cyclohydrolase was lower and was calculated to be 121 min. Phosphate buffers (1 0 - 100 mM) had no modulating effect on thc activity of GTP cyclohydrolase 1 (data not shown). ~

100

200

300 4 0 0

500

600

IS1 ( Y M)

Fig. 3. Basic kinetic properties of GTP cyclohydrolase 1, purified by affinity chromatography on GTP-agarose (a) and UTP-agarose (b). The incubation mixture contained 0.05-2.5 mM GTP and enzyme (< 200 nM) in 25 mM Tris/HCl, pH 8.0. The reaction was performed as described in the Materials and Methods. The effect or KCl (c) was studied using UTP- agarose-purified enzyme and the standard reaction system as dcscribed for (a) and (b).

Reaction kinetics of GTP cyclohydrolase I The basic kinetic properties of GTP-cyclohydrolase-I preparations from GTP-agarose and UTP-agarosc are shown in Fig. 3. Both preparations show a flat plot of V , versus substrate concentration, indicating that the enzyme is rather

'

~

'

Effect of divalent cations on GTP-cyclohydrolase-I activity Contrary to previous reports [2 - 61 there was no inhibition of GTP cyclohydrolase I by divalent cations. There was a clear positive effect of MgC12 on the activity of the UTP-purified GTP cyclohydrolase I, with an optimum concentration of 8 mM Mg2+ (Fig. 4). Since 0.1 M KC1 is present in the incubation buffer, this effect occurs in addition to the modulation by KCI and is the same for incubation with phosphate buffers (data not shown). When dihydroneopterin triphosphate and GTP were determined simultaneously, the sum of substrate consumed and product formed was always constant (Fig. 4).

565

8 ._

E

\

20 min

!L

L

0 a

500

% I z ..-. 0 8

0 ._

L

m

c

LL 0

b

100

2

10

18

26

[Mg2$mM)

Fig.6. Effect of chelate complexes of divalent cations on GTPcyclohydrolase-I activity. The reaction was carried out using GTP agarose-purified enzyme. The incubation buffer contained 0.1 M KCl and 1 mM EDTA. UTP was incubated with the enzyme for 5 min. NH2TP, dihydroneopterin triphosphate. ( 0 ) .with 1 mM EDTA, without UTP; (A),with 1 mM EDl'A and 1 1 pM UTP.

fect of the chelate complexes, depending on the concentration of M g Z f .This effect can be counteracted by addition of UTP to the reaction mixture. At a concentration equimolar to the enzyme, UTP totally abolished the inhibition by chelate complexes (Fig. 6, GTP-affinity-purified enzyme). Effect of tetrahydrobiopterinon GTP-cyclohydrolase-I activity

0

5

Retention Time, min

Fig.5. HPLC analysis of the calcium-dependent formation of a new product by GTP cyclohydrolase 1. (a) Standard neoplerin lriphosphate; (b-d) products formed in the presence of 16 mM Ca". The reaction conditions were as described for effector studies (see Materials and Methods) using UTP-purified enzyme. Oxidized samples were analyzed by HPLC on a Partisil-10 SAX anion-exchange column using fluorimetric detection. The maximum formation of the new product was observed after 60 min. (*), unknown product.

The activity of both GTP-purified and UTP-purified GTP cyclohydrolase I was inhibited by approximately 50% at a tetrahydrobiopterin concentration of 100 pM. However, the absolute activity or turnover of the UTP-purified enzyme was always higher (Fig. 7a). Therefore the influence of UTP on the inhibitory effect of tetrahydrobiopterin was tested. As shown in Fig. 7 b, addition of UTP to GTP - agarose-purified enzyme in a concentration equimolar to the enzyme subunits did not suppress the inhibition by tetrahydrobiopterin. Although the enzyme was inhibited in the presence of UTP, the activity was modulated to a higher level. The same effect was observed when tetrahydrobiopterin substituted at position 7 was used for the inhibition studies (data not shown). DISCUSSION

When GTP cyclohydrolase I was incubated in the presence of Ca2+ and neopterin triphosphate measured, a new product was formed depending on the Ca2' concentration and the incubation time. Fig. 5 shows the formation of the unknown product by the UTP-affinity-purified enzyme in the presence of 16 mM Ca2 . GTP-affinity-purified enzyme already yields the new product at 8 mM Ca2+ (data not shown). +

Effect of chelate complexes When 1 mM EDTA was added to the incubation mixture containing divalent cations, there was a strong inhibitory ef-

Data obtained from enzymic studies of GTP cyclohydrolase I so far have been controversial for the following reasons. The instability of the eukaryotic cnzyme renders kinetic experiments using highly purified enLyme impossible ; activity in crude tissue extracts is usually low and difficult to determine due to the presence of interfering substances; the assay methods used are different so that results can not always be compared [ 2 8 201. Some characteristics of both the prokaryotic and eukaryotic GTP cyclohydrolasc I provide evidence that it might be a regulatory enzyme: location at the first step of a ~

566 E. c a l f

R a t 1 iv e r

Lys Ala T h r Val A l a

142

A r g Val His I l e G l y

Fig. 8. Local similarity of the amino acid sequence of E. coli and rat GTP cyclohydrolase I. The 30-residue internal sequence of GTP cyclohydrolase I from E. coli was obtained after CNBr cleavage.

400

800

1200 1600

[Wj (PM) Fig. 7. Influence of tetrahydrobiopterin on the reactivity of GTP cyclohydrolase 1. Enzyme purified either with GTP-agarose (a) or UTP-agarose (A j was incubated with different concentrations of tetrahydrobiopterin (a). When the GTP- agarose-purified enzyme received prior incubation with UTP for 5 min, the inhbitory effect of tetrahydrobiopterin (BH,) was diminished (b); (a),without UTP, (A),with 11 pM UTP. NH2TI’, dihydroneopterin triphosphate.

multienzyme biosynthetic pathway; its unique protein structure. The GTP cyclohydrolases characterized so far are all large enzyme complexes which differ in size and number of subunits. In this respect, the GTP cyclohydrolases from E. coli (molecular mass of complex form 210 kDa, subunits 25 kDa) [3], D. melunoguster (complex form 575 kDa, subunits 39 kDa} [4], human liver (complex form 440 kDa, subunits 50 kDa} [5] and rat liver (complex form 300 kDa, subunits 30 kDa) [ti], are corresponding enzymes. In the present study the homopolymeric structure of GTP cyclohydrolase was clearly demonstrated for the E. coli enzyme. ldentical N-terminal sequences up to 50 residues were obtained from the homogeneous enzyme preparation as well as from the subunits blotted on polyvinylidene difluoride membrane. From this result and from the fact that the abovementioned corresponding enzymes show single bands in SDS/

PAGE, GTP cyclohydrolases of these species may also be homopolymers. This does not necessarily mean similarity in amino acid sequence. There is an obvious sequence similarity of 28% in the 50 N-terminal amino acid residues between the E. coli enzyme and the recently cloned rat liver GTP cyclohydrolase I [21], which do not greatly differ in subunit molecular mass (see above). Within the 30-residue internal sequence obtained after chemical cleavage with CNBr we identified a short stretch of 5 residues almost identical to a 5residue sequence (amino acid 133- 137) in the rat liver enzyme : Glu-His-His-Phe-Val (E. coli) and Glu-His-His-LeuVal (rat). In addition, the methionine residue that we cleaved is identified as Met120 in the rat enzyme. and the 30 following residues show 42% similarity between E. coli and rat liver GTP cyclohydrolase (Fig. 8). The recently published deduced amino acid sequence of E. coli GTP cyclohydtolase I [7] confirms our findings and adds further data to the fact that highly conserved sequences most probably related to the specificity of the enzyme are located in the middle or C-terminal region. In particular, the similarity of the internal 30-residue sequences of the two evolutionary different species mentioned above makes it obvious that these regions may also be homologous in other GTP cyclohydrolases. Furthermore, this sequence has not been detected in any other proteins so far registered in the EMBL Data Bank. The N-terminal half of the enzyme seems more variablc, containing bulky regions which contribute to the great differences in the molecular masses of GTP cyclohydrolases from different species. The use of two well-defined monoclonal antibodies, antibody l F l l related to the substrate-binding site [S], and the anti-idiotypic antibody NS7 [17] related to the pterin-binding site, as probes in Western-blot analysis strongly supported the allostery of GTP cyclohydrolase I. Since GTP, but not tetrahydrobiopterin, prevented binding of 1F1 1, and since binding of NS7 was abolished by tetrahydrobiopterin but not by GTP (see Table l), these ligands must have two different binding sites on the enzyme subunit. That means that tetrahydrobiopterin does not bind competitively to the GTPcyclohydrolase substrate site [8] but to another, regulatory, binding site. The findings that in the presence of UTP, binding of antibody NS7 is totally abolished, and that UTP does

567 not compete with GTP (see Table l), suggest that the two nucleotides do not share the same binding site on the enzyme subunit. Binding of UTP to a third site induces conformational changes in the antigen, preventing the binding of the antibody molecule. We can conclude that the binding sites for GTP, UTP and tetrahydrobiopterin must be separate sites, the latter two probably inducing conformational changes in the tertiary protein structure of the enzyme. Systematic enzymological investigation of the effect of UTP, which in our hands was not a competitive inhibitor, and other modulator molecules confirmed the allosteric characteristics of GTP cyclohydrolase observed in the immunochemical study. Wc could not observe Michaelis-Menten behaviour, nor sigmoid kinetics, indicating positive cooperativity as described for the rat enzyme [6]. The flat hyperbola obtained (Fig. 3 a), the slow response to substrate changes, and lhe low turnover number for GTP indicate a negative cooperativity according to the model described by Koshland [22] and Pauling [23]. Thus binding of GTP to one subunit induces the conversion from the low-affinity conformation to the high-affinity form, while the conformation of the other subunits remains unchanged. The transition phase occurring in the kinetic curve of UTPpurified GTP cyclohydrolase (Fig. 3 b) indicates that therc is still UTP bound to the enzyme. The binding of UTP affects the substrate-induced transformation of the enzyme subunits into the high-affinity form in such a way that at a certain GTP concentration the high-affinity state is reached faster. This characteristic of allostery is described as hysteretic enzyme kinetics [24]. It explains the higher turnover number and higher specific activity of the UTP-purified cyclohydrolase in spite of being as pure as the GTP-purified enzyme. Furthermore, the negative cooperativity makes GTP cyclohydrolase insensitive to minor changes of GTP, which is always present in the cells in high concentrations. In resting blood cells GTP concentrations are between 100-200 pM/l [25],which is in the range of the K, for GTP cyclohydrolase. An increase in enzymic activity can therefore only be achieved by a drastic increase of the GTP concentration, as it occurs in activated cells [9]. The difference between the GTP - agarose-purified and UTP agarose-purified enzyme is probably not on the protein levcl, since N-terminal and internal sequences are identical. The difference is rather due to the tight binding of an effector molecule, UTP, at a site other than the substrate-binding site. The kinetic curve (Fig. 3 b) shows that UTP cannot be displaced from the enzyme by high GTP concenlrations, yielding a change in the kinetic properties. In contrast, when UTP is added to the GTP-agarose preparation the kinetics of this enzyme preparation change (Fig. 6). The modulation of GTP-cyclohydrolase activity by KC1 is consistent with earlier reports [2 -61. The allosteric modulation of UTP is obvious in connection with the effects of other low-molecular-mass substances. In contrast to earlier results, we found no inhibition of GTP cyclohydrolase I by divalent cations. As shown in Fig. 4, MgClz enhanced the activity of UTP-purified cyclohydrolase, with an optimum concentration of 8 mM Mg2+.When EDTA is added in the presence of divalent cations, a strong inhibition can be observed depending on the concentration of cations (Fig. 6). Since EDTA alone had no eflect on cyclohydrolase activity, the inhibition must be caused by the chelate complexes formed and is equal to either UTP-purified or GTP-purified enzyme. The finding that addition of UTP in ~

equimolar amounts to the enzyme subunits totally abolished the inhibition by chelate complexes further supports the allosteric mechanism of regulation. The conformational changes induced upon binding of UTP to each subunit makes the attachment of the chelate complexes impossible. When GTP cyclohydrolase I was incubated in thc presence of CaClz and neopterin triphosphate was measured, a new product was formed (Fig. 5). Since the enzyme preparation is absolutely pure, this must be an as yet unexplained effect of Ca2?.Comparison of the retention time of the new compound with that of synthetic cyclic neopterin monophosphate suggests that the compound could be a neopterin monophosphate derivative. The finding that tetrahydrobiopterin, the metabolic end product in organisms other than E. coli, clearly inhibited E. coli cyclohydrolase, suggests that all GTP cyclohydrolases are regulated by a common mechanism. At a tetrahydrobiopterin concentration of 100 ~LM, GTP-cyclohydrolase activity was inhibited by 50%. UTP modulated the activity to a higher level although it did not suppress the inhibition by tetrahydrobiopterin (Fig. 7 b). We observed the same effect also with tetrahydrobiopterin substituted at position 7 (data not shown). Since all the data discussed above were obtained with absolutely pure enzyme, the apparently complex kinetic properties described are characteristic for GTP cyclohydrolase I. Indeed, the enzyme combines two different kinetic mechanisms as suggested in earlier work [4]: The enzyme’s activity can be modulated positively (cooperative) and negatively (non-cooperative) by allosteric effector molecules. GTP cyclohydrolase can therefore be classified as an M-type allosteric enzyme [26]. The authors thank M. Killen for assistance in manuscript preparation. This work was supported by a grant from the Swiss National Scicnce Foundation, project 31-33897.92.

REFERENCES 1. Kaufman, S. (1974) in Aromatic amino acids in the bruin. pp. 85 115, Elsevier, New York. 2. Blau, N. & Niederwieser, A. (1985) J . Clin. Chem. Clin.Biochem. 23, 169-174. 3. Yim, J. J. &Brown, G. M. (1976) J . Biol. Chem.251,5087-5094. 4. Weisberg, E. P. & O’Donnell, J. M. (1986) J . Bid. Chem. 261, 1453- 1458. 5. Schoedon, G., Rcdweik, U. & Curtius, H.-Ch. (1989) Eur. J . Biochem. 178, 627 634. 6. Hatckayama, K., Harada, T., Sumki, S., Watanabe. Y. & Kagamiyama, H. (1989) J. Biol. Chnn. 264, 21 660-21 664. 7. Katzenmeier, G., Schmid, C., Kellermann. J., Lottspeich, F. & Racher, A. (1991j Bid. Chem. Hoppe-Seyler 372. 991 -997. 8. Bellahsene, Z.,Dhondt, J. L. & Farriaux, J. P.(1984) Biochem. .I. 217.59-65. 9. Schoedon, G.: Troppmair, J., Fontana, A,, Huber. Ch., Curtius, 11.-Ch. & Niederwieser, A. (1987) Eur. J . Biochem. 166, 303 310. 10. Abou-Donia, M. M., Duch, D. S . , Nichol, C. A . & Viveros, 0. H. (1983) Endocrinology 112,2088-2094. 11. McLean. J. K.,Boswell, R. & O’Donnell, J. M. (1990) Genetics 126, 1007- 101 9. 12. Blau, N., Niederwieser, A., Redweik. U., Schoedon. G. & Leimbacher, W. (1989) in Pteridines and hiogenic amines in neuropsychiatry, pediatrics and immunology (Ixvine, R . A., Milstien, S., Kuhn, D. M., Curtius, H.-Ch., eds) pp. 97-105, Lakeshore Publishing Company, Grosse Pointe. 13. Ferre, J., Yim, J. J. & Jacobsen, K. B.(1986) J. Clzramutogr. 357, 283 - 292. ~

568 14. Laemmli, U. K . (1970) N f f f w e2.27; 680-685. 15. Frank, G. (1989) in Methods inprotein sequence unulysis.proceedings of the 7th international conference (Wittmann-Liebold, B., ed.) pp. 116- 121, Springer-Verlag, Berlin. 16. Towbin, H., Staehclin, Th. & Gordon, J.(1979) Proc. Nutl Acnd. Sci. U S A 76, 4350-4354. 17. Jennings, J. &Cotton, K. G. H. (1990) .I. Biol. Chem. 265,18851889. 1R. Burg, A . W. & Brown, G. M.(1968) J . Biol. Chem. 243, 23492358. 19. Duch, D. S., Bowcrs, S. W., Woolf, J. H. & Nichol, C. A. (1984) Life Sci.35, 1895 - 1901. 20. Blau, N. & Niederwieser, A. (1Y83) A n d . Biochem. 128, 446452.

21. Hatekayama, K., Tnoue, Y . , Harada, T. & Kagamiyama, H. (199 1) J. Bid. Chem. 266,765 769. 22. Koshland, D. E. Jr (1970) in The enzymes (Boyer, P. D., ed.) vol. 1, pp. 341 -396, Academic Press, New York. 23. Pauling, L. (1935) Proc. Natl Acad. Sci. U S A 2 1 , 186-191. 24. Frieden, C. (1970) J . Biol. Chem. 245, 5788 - 5799. 25. Simmonds, H. A,, Dulley, J. A. & navies, P. M. (1991) in Techniques in diagnostic human hiochemicnl genetics (Hommes, F. A., ed.) pp. 397-424, Wiley-Liss Inc., New York. 26. Lehninger, A. L. (1Y77) in Biochemistr), (Lehningcr, A. L.. ed.) pp. 217-248, Worth Publishers Inc., New York. -