Eur. J. Biochem. 80, 349-357 (1977)

Mechanism of the Prolyl Hydroxylase Reaction 2. Kinetic Analysis of the Reaction Sequence

Raili MYLLYLA, Leena TUDERMAN, and Kari I. KIVIRIKKO

Department of Medical Biochemistry, University of Oulu

(Received December 13, 1976iJuly 25, 1977)

The kinetics of the prolyl hydroxylase reaction were studied with pure enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs of Fe2+, 2-oxoglutarate, 0, and the polypeptide substrate, whereas parallel lines were obtained for pairs involving ascorbate with each substrate. In addition, parallel lines were obtained when the polypeptide substrate concen- tration was varied at different fixed 2-oxoglutarate concentrations in the presence of saturating 0, concentration. Poly(L-proline) was a competitive inhibitor with respect to the polypeptide substrate, but uncompetitive with respect to Fe2+ and 2-oxoglutarate. High concentrations of the polypeptide substrate inhibited the reaction, this substrate inhibition being competitive with respect to Fe2+ and 2-oxoglutarate. Succinate, CO, and collagen were product inhibitors, succinate inhibiting the reaction competitively with respect to 2-oxoglutarate, but noncompetitively with respect to the other substrates, and collagen noncompetitively with respect to all substrates. The apparent K, and Ks values for the substrates and Ki values for the inhibitors are given.

These and additional data would be consistent with a tentative reaction scheme involving an ordered binding of Fe2+, 2-oxoglutarate, 0, and the polypeptide substrate to the enzyme in this order, the binding of Fe2+ being at thermodynamic equilibrium. The enzyme can also react directly with the polypeptide substrate or its analogue poly(L-proline) under certain conditions, forming dead-end complexes. The products are released only after the hydroxylation, possibly in the order : the hydroxylated polypeptide, CO, and succinate. Ascorbate may react either with enzyme.Fe before the release of Fe2+ or with free enzyme before the binding of Fe2+, but a reaction with ascorbate at any stage after the release of the first product is not excluded. The mechanism proposed is not entirely identical with either of the main two previous suggestions for the mechanism of 2-oxoglutarate dioxygenases.

Prolyl hydroxylase belongs to the group of 2-0x0- glutarate dioxygenases, other members of which in- clude lysyl hydroxylase, 4-butyrobetaine hydroxylase, thymine 7-hydroxylase, pyrimidine deoxyribonucleo- side 2'-hydroxylase and p-hydroxyphenylpyruvate hy- droxylase (for reviews, see [l -41). These enzymes require molecular oxygen, ferrous iron, 2-oxoglutarate and a reducing agent. p-Hydroxyphenylpyruvate hy- droxylase, the substrate of which contains an 0x0 group, differs from the other enzymes in that it does not need 2-oxoglutarate but has apparently a similar reaction mechanism. The 2-oxoglutarate is stoichio- metrically decarboxylated during these reactions [ 1 - 61,

Abbreviation. (Pro-Pro-Gly), , a linear copolymer composed of regular alternating sequences of L-proline-L-proline-glycine.

Enzyme. Prolyl hydroxylase or prolyl-glycyl-peptide,2-0~0- glutarate: oxygen oxidoreductase (4-hydroxylating) (EC 1.14.1 1.2).

and one atom of the oxygen molecule is incorporated into the hydroxyl group while the other is incorporated into the succinate [7,8].

Two main possibilities have been considered for the reaction mechanisms of these enzymes [l -41. In studies on 4-butyrobetaine hydroxylase, Lindstedt and co-workers [9] suggested that the reaction might involve a complex between the enzyme, Fez+, 0, and the substrate, with subsequent formation of a peroxide intermediate of the substrate. This peroxide would then make a nucleophilic attack on the carbonyl car- bon of the 2-oxoglutarate forming a peroxide bridge between the two compounds, and finally this inter- mediate would be decomposed to the hydroxylated product, succinate and CO, . However, Hamilton [lo, 111 favours an oxenoid mechanism, since the sub- strates of all these enzymes are simple aliphatic com-

350 Mechanism of Prolyl Hydroxylase

pounds, and formation of the peroxide intermediate would be a difficult reaction. In this scheme, 0, com- plexed to iron initially attacks the 2-oxoglutarate, forming a persuccinic acid intermediate and CO, . The persuccinic acid then reacts with the substrate pro- ducing the hydroxylated product and the succinate. Some evidence has been presented for and against both mechanisms [I -41 but neither of them has yet been proved.

One possible approach to this problem would be to carry out a kinetic analysis of the reaction sequence. An initial attempt to elucidate the kinetics of prolyl hydroxylase has been reported [12], but the experi- ments were performed with an impure enzyme and with a relatively insensitive and tedious assay proce- dure. Only a limited number of different experiments could be carried out, and the data were difficult to interpret. In the present study, an extensive analysis was carried out with respect to initial velocity, sub- strate inhibition and product inhibition kinetics, and to the kinetics of inhibition with poly(L-proline), a competitive inhibitor with respect to the polypeptide substrate. On the basis of these and other data [13], a reaction mechanism is proposed which is not entirely indentical with either of the two previously suggested [9-111.

MATERIALS AND METHODS

Materials

As described in the preceding paper [13], entirely pure prolyl hydroxylase from chick embryos was used as the enzyme. The sources of the other materials are also given in the preceding paper [13]. Additional materials were glomerular basement membrane col- lagen from human kidney [14], and citrate-soluble collagen from rat skin [15] which were prepared as reported elsewhere.

Assay of Prolyl Hydroxylase Activity

The enzyme activity was assayed under standard conditions in a final volume of 1 .O ml which contained 0.05-0.2 pg enzyme, 0.5 mg (Pro-Pro-Gly), . 4H,O or 0.1 mg (Pro-Pro-Gly),, . 9H,O, 0.05 pmol FeSO,, 0.1 pmol 2-oxo[l -14C]glutarate (40000 dis. . min-I), 1 pmol ascorbate, 0.1 mg catalase, 0.1 pmol dithio- threitol, 2 mg bovine serum albumin and 50 pmol Tris-HC1 buffer adjusted to pH 7.8 at 25 "C, as de- scribed in detail in the preceding paper [13]. The reaction took place at 37 "C for 30 min, and the I4CO, formed was trapped and counted [13].

In the experiments, in which the 0, or CO, con- centration was varied the gas mixture were prepared and the solution equilibrated as described elsewhere [13]. In the experiments with CO,, appropriate con-

centrations of NaHCO, were also added to prevent changes in the pH [16].

RESULTS

INITIAL VELOCITY STUDIES

The prolyl-hydroxylation-coupled decarboxylation of 2-oxoglutarate under the conditions used in this study was linear with time and enzyme concentration. In all the experiments reported below the formation of I4CO2 corresponded to a hydroxylation of less than 3% of the prolyl residues in the positions preceding the glycyl residues of the polypeptide substrate, only these prolyl residues being hydroxylated by the enzyme [17,18]. Detailed kinetic studies were carried out by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other sub- strates were held constant.

Since recent kinetic studies on other enzymes have indicated that trace amounts of impurities present in commercial metal salt preparations can affect the results [19,20], the experiments in which the Fe2+ concentration was varied were carried out with FeSO, purified with dithizone [13]. Although no evidence for the chelation of Fe2+ with the Tris buffer was observed, the experiments reported in Fig. 1 A-C (below) were repeated with 50 mM 3-(N-morpholino)-propanesul- fonate buffer with a negligible metal-binding tendency [21]. The results were indentical with those shown in Fig. 1 A-C. Significant binding of Fe2+ to 2-0x0- glutarate and ascorbate seems unlikely, due to the low concentrations of these compounds. Attempts were also made to study the binding of Fe2+ to the poly- peptide substrate by ultrafiltration experiments, but the results were inconclusive as considerable amounts of Fe2+ became bound to the ultrafiltration membrane even in the absence of the polypeptide substrate. The free Fez+ concentrations could thus not be corrected for possible binding of part of Fe2+ to the polypeptide substrate.

When the initial velocity of the reaction was studied by varying the Fe2+ concentration at different fixed concentrations of 2-oxoglutarate and at constant con- centrations of the other components, the double- reciprocal plots intersected to the left of the ordinate (Fig. lA), whereas when the data were plotted as (velocity) versus [2-oxoglutarate] at different fixed concentrations of Fe2 + and at constant concentrations of the other components, the lines intersected on the ordinate (Fig. 1B). The slopes of the lines obtained in Fig. 1 A, when plotted against [2-oxoglutarate] gave a line which passed through the origin (Fig. 1C).

When the Fe2+ concentration was varied at dif- ferent fixed concentrations of the polypeptide sub- strate (Fig. 2) or 0, (data not shown) and at constant

R. Myllyll, L. Tuderman, and K. I. Kivirikko 351

4t t

l / [ F e 2 + ] (rnM-')

Fig. 2. Effect ofFe'+ concentration on the rate ofprolyl hydroxylase reaction at different fixed concentrations of (Pro-Pro-Gly), and constant concentrations of the other components. The concentrations of the polypeptide substrate were: (0) 0.1 mg/ml, (0) 0.2 mg/ml, (0) 0.3 mg/ml, (m) 0.4 mg/ml. The concentration of 2-oxoglutarate was 0.1 mM, ascorbate 1 mM and 0, 200 pM. u was measured as dis. . min-'

15

1 . - 0. 10 0 -

5

0 -0250 -0125 0 0125 0250 0375 0500

1 / [ 2-0xoglutarate] (rnM-')

t C Fig. 3. Effect of F2' concentration on the rate of prolyl hydroxylase reaction at different fixed concentrations of ascorbate and constant concentrations of the other components. The concentrations of as- corbate were: (0) 0.125 mM, (0) 0.250 mM, (0) 0.375 mM, (B) 0.500 mM. The concentration of 2-oxoglutarate was 0.1 mM, (Pro-Pro-Gly),, 0.1 mg/ml and 0, 200 pM. u was measured as dis. . min-'

concentrations of the other components, the double- reciprocal plots intersected to the left of the ordinate. Similarly when the polypeptide substrate or 0, con- centration was varied at different fixed concentrations of Fe2+ and at constant concentrations of the other components, the double-reciprocal plots intersected to the left of the ordinate (data not shown). By contrast, parallel lines were obtained when the Fe2+ concen- tration was varied at different fixed ascorbate con- centrations and at constant concentrations of the other components (Fig. 3).

Lines intersecting to the left of the ordinate were obtained when the 2-oxoglutarate concentration was varied at different fixed concentrations of the poly- peptide substrate (Fig. 4) or oxygen (data not shown)

0 I V I I I I -0250 -0125 0 0725 0250 0375 0500

l / [2 -0xog lu ta ra t e ] (mM- ' )

Fig. 1. Effect of Fe2+ concentration on the rate ofprolyl hydroxylase reaction at different $xed concentrations of 2-oxoglutarate ( A ) and the effect of 2-oxoglutarate concentration on the rate of prolyl hydroxylase reaction at different fixed concentrations of Fez' ( B ) . (A) The concentrations of 2-oxoglutarate were: (0) 0.02 mM, (0) 0.04 mM, (0) 0.06 mM, (H) 0.08 mM, (B) The concentrations of Fez+ were: (0) 0.0025 mM, (0) 0.0050 mM, (0) 0.0100 mM, (B) 0.0175 mM, (0) 0.025 mM. The concentrations of the other components were constant: (Pro-Pro-Gly),, 0.1 mg/ml, ascorbate 1 mM and 0, 200 pM. (C) A secondary plot of the slopes of the lines obtained in A when plotted against [2-oxoglutarate]-'. u was measured as dis. . m1n-l

352

I

Mechanism of Prolyl Hydroxylase

I I I

l/ [2 -0xog lu ta ra te ] ( rnM- ' )

Fig. 4. Effect of 2-oxoglutarate concentration on the rate of prolyl hydroxylase reaction at different $xed concentrations of (Pro-Pro- Gly), and constant concentrations of the other components. The concentrations of the polypeptide substrate were: (0) 0.1 mg/ml, (0) 0.2 mg/ml, (0) 0.3 mg/ml. The concentration of Fe2+ was 0.05 mM, ascorbate 1 mM and 0, 200 pM, u was measured as dis. . min

and with constant concentrations of the other com- ponents. Similar intersecting lines were also obtained when the polypeptide substrate concentration was varied at different fixed concentrations of 0, (Fig. 5) or 2-oxoglutarate (data not shown) and at constant concentrations of the other components. However, parallel lines were obtained, when the polypeptide substrate concentration was varied at different fixed 2-oxoglutarate concentrations in the presence of saturating O2 concentration and at constant concen- trations of the other components (Fig. 6). Parallel lines were also obtained when the ascorbate concen- tration was varied at different fixed concentrations of 2-oxoglutarate (data not shown), 0, (data not shown) or the polypeptide substrate (Fig. 7).

-

5 . - 0

0

-004 -002 0 0 0 2 004 006 008 010 l / [ ( P r ~ - P r o - G l y ) , ~ ] ( r n l i r n g )

Fig. 6. Effect of (Pro-Pro-Gly),, concentration on the rate of prolyl hydroxylase reaction at different Jixed concentrations of 2-oxogluta- rate at saturating 0, concentration (1000 p M ) and at constant concentrations of the other components. The concentrations of 2-0x0- glutarate were (0) 0.01 mM, (0) 0.015 mM, (0) 0.025 mM, (m) 0.045 mM. The concentration of Fe2+ was 0.05 mM and ascorbate 1 mM. c was measured as dis. . min-'

In summary, intersecting lines in double-reciprocal plots were given by all possible pairs involving Fe2+, 2-oxoglutarate, 0, and the polypeptide substrate, whereas parallel lines were obtained for pairs com- prising ascorbate and each of the other substrates. In addition, parallel lines were obtained when the poly- peptide substrate concentration was varied at different fixed 2-oxoglutarate concentrations in the presence of saturating 0, concentration. The apparent K, and K, values for the substrates are shown in Table 1. A good agreement was found between the values obtained in the different experiments. K, values have been studied previously for less purified enzyme preparations [ 12, 17,22,23]. All present values agreed within about a factor of two with those reported previously. The dis- sociation constants were determined for the first time and were found to differ only slightly from the K, values (Table 1).

INHIBITION STUDIES

Inhibition by Poly (L-proline)

Poly(L-proline) has previously been reported to act as a competitive inhibitor of prolyl hydroxylase with respect to the polypeptide substrate [16,24,25]. The present experiments confirmed that inhibition by poly(L-proline) was competitive with respect to the polypeptide substrate (Fig. 8A), whereas it was un- competitive with respect to Fe2+ (Fig. 8B) and 2-0x0- glutarate (data not shown).

Substrate Inhibition

A high concentration of the polypeptide led to obvious substrate inhibition, as shown in Fig. 9A for (Pro-Pro-Gly),, . With this substrate the inhibition

R. Myllyla, L. Tuderman, and K. I. Kivirikko 353

l / [ Ascorbate] (rnM-')

Fig. 7. Effect of ascorbate concentration on the rate of prolyl hydroxy- lase reaction at different fixed concentrations of (Pro-Pro-Gly),, and constant concentrations ofthe other components. The concentra- tions of polypeptide substrate were: (0) 0.01 mg/ml, (0) 0.02 mg/ml, (0) 0.03 mg/ml, (m) 0.04 mg/ml. The concentration of Fe2+ was 0.05 mM, 2-oxoglutarate 0.1 mM and 0, 200 pM. v was measured as dis. . min-'

2 . - 0

0

1

C

t A

10 -005 0 005 010 015 020 l / [ ( Pro- Pro-Gly ),I (m l /mg)

c

l / [ F e 2 + ] (mM-') Fig. 8. Inhibition oftheprolyl hydroxylase reaction by poly(L-proline) with respect to (Pro-Pro-Gly), ( A ) or Fez+ ( B ) . The concentrations of poly(L-proline) were: A: (0) 3.75 pg/ml, (0) 2.5 pg/ml, (0) 1.25 pg/ml, (m) none, B: (0) 2.5 pg/ml, (0) 2.0 pg/ml, (0) 1.0 d m l , (m) none. The concentration of Fe2+ was 0.05 mM (A), (Pro-Pro- Gly), 0.5 mg/ml (B), 2-oxoglutarate 0.1 mM, ascorbate 1 mM and 0 2 200 pM. u was measured as dis. . min-'

Table 1. Apparent kinetic constants for the substrates of the prolyl hydroxylase reaction The apparent kinetic constants were determined from data obtained in various experiments on initial velocity studies. The values shown do not represent true constants as the concentrations of only two substrates were varied in each experiment. K,- K, are Michaelis constants and Ki, - Kid dissociation constants. The values were determined from primary plots according to the equation

for intersecting lines or

for parallel lines, and from secondary plots of intercepts and slopes of the primary plots versus [fixed substratel-' [29]. The values were not corrected for the possible binding of part of Fez+ to the poly- peptide substrate

Substrate Kinetic Apparent Obtained constant value from Fig. no.

Fez+ (A) K, 5.2 2 Ka 2.8 3 Kia 5.2 2 Kia 5.0 not shown

2-Oxoglutarate (B) Kb 22 1 A Kb 22 4 Ki b 19 4

not shown Kib 19

0 2 (C) Kc 40 5

Ki c 40 5 Kc 45 not shown

(Pro-Pro-Gly), (D) Kd Kd Kd

Kid Kid

(Pro-Pro-Gly),, (D') Kd, Kd '

Kd. Kid

w/ml 180 280 250 170 180

35 41 42 25

4 not shown 2 not shown 4

not shown 5 7 5

PM Ascorbate" (E) Ke 300 3

Ke 300 7

a Ascorbate is not stoichiometrically consumed in the reaction ~ 3 1 .

took place when the concentration exceeded about 200 pg/ml, whereas higher concentrations were re- quired for substrate inhibition with (Pro-Pro-Gly), . The substrate inhibition was competitive with respect to Fez+ (not shown) and 2-oxoglutarate (Fig. 9B).

Product Inhibition

Succinate was found to be a product inhibitor of the reaction. The inhibition was competitive with

354 Mechanism of Prolyl Hydroxylase

0 0.2 04 0 6 l / [ ( P r o - P r ~ - G l y ) , ~ ] ( rn l i rng)

1 / [2-0xoglutarate] (mM-')

Fig. 9. Substrate inhibition of the prolyl hydroxyluse reaction by (Pro-Pro-Gly),, . (A) Effect of increasing substrate concentration. (B) Inhibition of the reaction with respect to 2-oxoglutarate. The concentrations of (Pro-Pro-Gly),, were (B): (0) 1.6 mg/ml, (0) 1.2 mg/ml, (0) 0.8 mg/ml, (m) 0.2 mg/ml. The concentration of Fez+ was 0.05 mM, 2-oxoglutarate 0.1 mM (A), ascorbate 1 mM and 0 2 200 pM. u was measured as dis. . min-'

respect to 2-oxoglutarate (Fig. lOA), but noncom- petitive with respect to the other substrates (as shown in Fig. 10B with respect to the polypeptide substrate).

Attempts to study product inhibition with CO, were complicated, since this compound inhibited the trapping of the I4CO, formed in the reaction, and it was not possible to use the assay based on the measure- ment of the decarboxylation of 2-0x0 [ l-14C]glutarate. Therefore, the reaction was carried out with 0.1 mg (Pro-Pro-Gly),, as substrate, under standard con- ditions, except that unlabelled 2-oxoglutarate was used, and after hydrolysis with an equal volume of concentrated HCI at 120 "C for 16 h, the amount of hydroxyproline formed was assayed [26]. No inhibition was found with 1.2 or 2.4 mM CO, concentration, whereas 3.6 mM concentration inhibited the reaction by about 5%, 7.2 mM by 35% and 12 mM by 75%. Detailed kinetic studies were not carried out with respect to this inhibition, as in the presence of low

t A

- 4 - 2 0 2 4 6 8 10 l / [ ( P r ~ - P r o - G l y ) ~ ] ( rn l i r ng )

P

0 -02 - 0 1

l / [ 2-0xoglutarate] ( rnM- ' )

Fig. 10. Inhibition of theprolyl hydroxyluse reaction by succinate with respect to 2-oxoglutarate ( A ) and (Pro-Pro-Gly), ( B ) . The con- centrations of succinate were: A: (0) 5 mM, (.) 2.5 mM, (0) 1.25 mM, (m) none, B: (0) 5 mM, (0) 2.5 mM, (0) 1.25 mM (m) none. The concentration of Fez+ was 0.05 mM, 2-oxoglutarate 0.1 mM (B), (Pro-Pro-Gly), 0.5 mg/ml (A), ascorbate 1 mM and Oz 200 pM. v was measured as dis. . min ~'

polypeptide substrate concentrations it would be ne- cessary to assay accurately the formation of less than 10 nmol of hydroxyproline, and the assays are not sensitive and specific enough for such measurements in the presence of hydrolysis products of 2 mg of bovine serum albumin.

Collagen was also found to be a product inhibitor of the reaction. Since type IV collagen from basement membranes contains more hydroxyprolyl residues per a chain than type I collagen from a number of tissues such as skin, the glomerular basement membrane collagen and citrate-soluble rat skin collagen were compared as product inhibitors (Table 2). The base- ment membrane collagen was found to be a more effective inhibitor. Product inhibition with collagen was noncompetitive with respect to all substrates of the reaction (as shown in Fig. 11 with respect to the glomerular basement membrane collagen and poly- peptide substrate).

R. Myllyla, L. Tuderman, and K. I. Kivirikko 355

Table 2. Inhibition constants for poly(L-proline) and the product inhibitors The apparent Ki values werc calculated from the secondary plots. Ki, is the Ki calculated from the slopes and Kii the Ki calculated from the intercepts. Skin collagen used was citrate-soluble rat skin collagen, basement membrane collagen was human glomerular basement membrane collagen. CO, was also a product inhibitor, but detailed kinetics were not studied with respect to this inhibition (see text). When the inhibition was studied with 0.1 mg (Pro-Pro-Gly),,/ml, a 50% inhibition was found with about 10 mM concentration

Inhibitor 4% Kii

Poly(L-proline) M , 9000 2.5 2.3 Skin collagen 180 170 Basement membrane collagen 40 40

mM

Succinate 1.6 1.4

10

8

L

- 6

0 D

4

2

0

I / [ ( Pro-Pr~-Gly ) ,~ ) (rnl /mg)

Fig. 11. Inhibition of the prolyl hydroxylase reaction by basement membrane collagen with respect to the polypeptide substrate (Pro- Pro-Gly) The concentrations of basement membrane collagen were: (0) 15 pg/ml, (0) 10 pg/ml, (0) 5 pg/ml, (w) none. The con- centration of Fe2+ was 0.05 mM, 2-oxoglutarate 0.1 mM, ascorbate 1 mM and 0 2 200 pM. u was measured as dis. . min-'

Inhibit ion Constants

The apparent Ki values for inhibition with poly- (L-proline) and with the product inhibitors, as cal- culated from the secondary plots, are shown in Table 2.

DISCUSSION

Kinetic mechanisms for enzyme reactions fall into two major groups. In sequential mechanisms all the reactants must combine with the enzyme before the reaction can occur and any products can be released, whereas in substitution mechanisms one or more pro- ducts are released before all the substrates have be- come bound. Sequential mechanisms are called order-

ed if the reactants combine with the enzyme and dissociate in an obligatory order, or random if alter- nate pathways exist and the order of combination or release is not obligatory (see reviews [27 - 291).

In this study, intersecting lines were obtained in the double-reciprocal plots for all possible pairs in- volving Fez +, 2-oxoglutarate, 0, and the polypeptide substrate. This suggests that the binding of these sub- strates to the enzyme occurs by a sequential mechanism, either ordered or random [27-291. By contrast, parallel lines were obtained in the double-reciprocal plots for pairs comprising ascorbate and each of the other substrates. Accordingly, ascorbate should prob- ably be placed either as the first reactant in the reaction scheme or as a reactant combining with the enzyme only after one or more products have been released

Experiments in which the initial velocity of the reaction was studied by varying the Fez + concentration at different fixed concentrations of 2-oxoglutarate and at constant concentrations of the other components gave lines intersecting to the left of the ordinate, whereas when the data were plotted as (velocity)-' uersus [2-oxoglutarate] -' at different fixed concentra- tions of Fez+, the lines intersected on the ordinate, and when the slopes of the lines obtained in the former plotting were plotted against [2-oxoglutarate]-', a line was obtained which passed through the origin. These results indicate an ordered mechanism for the addition of Fez + and 2-oxoglutarate, with the addition of Fez+ being at the thermodynamic equilibrium [27-291. It thus follows that the addition of Fez+ occurs before that of 2-oxoglutarate, Fez+ cannot dissociate once 2-oxoglutarate has been added, and need not leave the enzyme during each catalytic cycle, but may remain bound to the enzyme [27 - 291.

Inhibition of the reaction by the analogue of the polypeptide substrate, poly(L-proline), was competitive with respect to (Pro-Pro-Gly), , but uncompetitive with respect to Fez+ and 2-oxoglurate. These findings would be consistent with an ordered binding of Fez+ and 2-oxoglutarate, these compounds becoming bound at an ealier stage than the polypeptide substrate [27 - 291. The finding that prolyl hydroxylase catalyzes decarboxylation of 2-oxoglutarate in the presence of Fez+ and 0, even in the absence of the polypeptide substrate [2,13] indicates that all these three reactants may become bound before the polypeptide substrate.

Product inhibition by collagen and succinate was noncompetitive with one single exception. This find- ing favours an ordered rather than a random mecha- nism [27 - 291. Variation of the polypeptide substrate concentration at different fixed 2-oxoglutarate con- centrations in the presence of saturating 0, concen- tration gave parallel lines. This observation suggests binding of 2-oxoglutarate, 0, and the polypeptide substrate in that order [28].

[27 - 291.

356 Mechanism of Prolyl Hydroxylase

Asc , Pept-OH co7 sy :f u , Fez. 1 ; I i ? Fe2' 4" t t * I I * I v

Fe2' 2 4 g 4 1 1 1

t "hpt

E EFe2' EFe2:2-Og EFe3'2-090; EFe3:2-0g0TPept EFe2:SuccC02 EFe2'Succ EFe" E EFe2'

E.Pept Fig. 12. Schematic presentation OJ the proposed mechanism for prolyl hydroxylase reaction. The dashed lines between the enzyme and Fez+ at the end of the cycle indicate that Fez+ need not leave the enzyme during each catalytic cycle. The dashed lines between the ascorbate and the enzyme indicate that ascorbate may react before or after the release of Fez+, but a reaction with ascorbate at any stage after the release of the first product is not excluded. It should be further noted that ascorbate is not stoichiometrically consumed in the reaction [13]. The 0, is shown here as being reduced to O;, although the precise form of the activated oxygen has not been elucidated [13]. Abbreviations: E = enzyme, 2-Og = 2-oxoglutarate, Pept =polypeptide substrate, Succ = succinate, Asc = ascorbate

Although the kinetic data thus indicate that the polypeptide substrate becomes bound after Fe2+ and 2-oxoglutarate, it has been clearly demonstrated that prolyl hydroxylase becomes bound in the absence of Fe2+ and 2-oxoglutarate to an affinity column con- sisting of a polypeptide substrate [30] or poly(L-pro- line) [31] linked to agarose. This disagreement is probably explained by the formation of a dead-end complex enzyme. polypeptide or enzyme. poly(L-pro- line). The formation of such dead-end products does not take place under normal reaction conditions. Evidence for this suggestion was obtained in the find- ing that the polypeptide in high concentrations acts as a substrate inhibitor and that this inhibition is competitive with respect to Fe2+ and 2-oxoglutarate. Since the binding of Fe2+ seems to be at thermodynamic equilibrium (see above), formation of a dead-end complex enzyme.polypeptide would be expected to cause such substrate inhibition [32].

Studies on product inhibition give initial informa- tion on the order of release of the products from the enzyme. The inhibition by succinate was competitive with respect to 2-oxoglutarate, but noncompetitive with respect to 0, and the polypeptide substrate. Accordingly, succinate reacts with a similar form of the enzyme as 2-oxoglutarate. Because Fe2+ is bound before 2-oxoglutarate (see above), succinate is prob- ably released before Fe2+ but after the hydroxylated polypeptide and CO, . Product inhibition with collagen was noncompetitive with respect to all the substrates of the reaction, and thus the hydroxylated polypeptide may be the first product to be released [29]. This sug- gestion would agree with a common feature of ordered reaction sequences in that the product of the last substrate to combine with the enzyme is the first to be released [32]. The present results would thus be consistent with the release of the hydroxylated poly- peptide, CO, and succinate in this order, prior to the release of Fe2+, but it should be noted that more complete studies on product inhibition will be neces- sary to verify the order of release of the products.

Ascorbate has not yet been placed in the reaction sequence. This compound is not stoichiometrically

consumed in the reaction [13], and thus ascorbate should probably not be considered as a true substrate. The kinetic data suggest that this compound does not react with the enzyme at any stage between the binding of Fe2+, 2-oxoglutarate, 0, and the polypeptide sub- strate. The exact role of ascorbate in the reaction is not known, although it has been regarded possible that this compound would be required to keep the enzyme or enzyme.Fe2+ in a reduced state [13]. In such case it would seem possible that ascorbate would react either with the enzyme.iron complex after suc- cinate has been released or with the free enzyme before Fe2+ becomes bound. Both these possibilities are consistent with the present kinetic data, but a reaction with ascorbate at any stage after the release of the first product is not excluded.

The data discussed above and in our preceding paper [13] are consistent with the reaction mechanism shown in Fig. 12. The enzyme reacts first with Fez+, this addition being at thermodynamic equilibrium. The second substrate binding is 2-oxoglutarate and the third 0,. At this stage Fe2+ is probably oxidized and 0, becomes activated, possibly to 0;. The activat- ed oxygen can catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the polypeptide substrate at a rate of about 4 mol of C0, formed (mol enzyme)-' min-'. However, in the normal reaction sequence the polypeptide substrate becomes bound before the uncoupled decarboxylation takes place, and the activated oxygen .2-oxoglutarate complex interacts with the prolyl residue forming a hydroxy- prolyl residue, CO, and succinate at a rate which is about 80 times higher than that of the uncoupled reaction. The products are released only after this reaction, possibly in the order : hydroxylated poly- peptide, CO, and succinate. The iron probably be- comes reduced back to Fe2+ when this reaction takes place, and the Fe2+ may subsequently be released from the enzyme, but it need not leave the enzyme be- tween each catalytic cycle. Ascorbate may react with the enzyme.iron complex before the release of Fez+ or with the free enzyme, to retain the enzyme.Fe2+ or free enzyme in its reduced state, but it should be noted

R. Myllyla, L. Tuderman, and K. I. Kivirikko 351

that a reaction with ascorbate at any stage after the release of the first product is not excluded. The enzyme can also form a dead-end complex with the polypep- tide substrate or its analogue poly(L-proline) under certain conditions, but the reaction does not continue from these dead-end products.

The reaction mechanism suggested by Lindstedt and co-workers [9] is not consistent with our kinetic data or with the uncoupled decarboxylation of 2-0x0- glutarate in the absence of the polypeptide substrate [2,13], as in their scheme the enzyme reacts with the substrate before the reaction with the 2-oxoglutarate. The mechanism suggested by Hamilton [lo] is not entirely in agreement with our scheme either, as the mechanism involves decarboxylation of 2-oxoglutarate to a persuccinic acid intermediate prior to the binding of the substrate. The release of CO, prior to the ad- dition of the polypeptide substrate is not consistent with our kinetic data.

Hobza et al. [33] studied the mechanism of the prolyl hydroxylase reaction using quantum chemical model calculations. They propose a third reaction mechanism which resembles that of the Hamilton mechanism, but in which the activated oxygen.2-0x0- glutarate complex reacts with the prolyl residue before the decarboxylation takes place. The reaction scheme they construct solely on the basis of the model cal- culations closely resembles our scheme, except that they suggest that ascorbate may be stoichiometrically consumed.

It should be noted that the kinetic analysis of en- zyme reactions has proved rather complicated in many instances, and the problem becomes even more dif- ficult when several reactants are involved [27 - 291. The present reaction has five reactants, and thus all the kinetic data must be interpreted with caution. The role of ascorbate and the order of release of the pro- ducts in particular require additional studies. Our reaction scheme should thus be regarded only as a tentative one which seems to be consistent with the data at present available. However, it is interesting to note that the main features of our scheme are in a good agreement with one constructed on the basis of model calculations [ 3 3 ] without any information on the kinetic data.

This work was supported in part by a grant from the Medical Research Council of the Academy of Finland. The authors grate- fully acknowledge the expert technical assistance of Miss Raija Leinonen and Mrs Lea Torvela.

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R. Myllyla, L. Tuderman, and K. I. Kivirikko, Oulun yliopiston laaketieteellisen kemian laitos, Kajaanintie 52A, SF-90220 Oulu 22, Finland