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Synthetic polypeptides as substrates and inhibitors of collagen proline hydroxylase
ARCHIVES
OF
BIOCHEMISTRY
Synthetic
AND
BIOPHYSICS
Polypeptides
779-785
(1968)
as Substrates Proline
J. J. HUTTON,
126,
JR., A. MARGLIN,
and
Inhibitors
of Collagen
Hydroxylase B. WITKOP,
J. KURT&
A. BERGER,
AND
S. UDENFRlEND Laboratory Inslitute
of Clinical of Arthritis Department
Biochemistry, and Metabolic of Biophysics,
National Heart Institute, and Laboratory of Chemistry, National Diseases, National Institutes of Health, Bethesda, Maryland; The Weizmann Institute of Science, Rehoooth, Israel Received
February
1, 1968
The synthetic polypeptide ([3,4-3H-Pro]-Gly-Pro), can be hydroxylated by collagen proline hydroxylase. The affinity of the enzyme for the substrate increases with increasing molecular weight of substrate over the range 1300-8000. The data indicate that maximal affinity should be observed with a polypeptide of molecular weight considerably greater than 8000. Poly-L-proline II and (Pro-Gly-Pro), appear to be COIV petitive inhibitors of the hydroxylation reaction. The series of oligopeptides, (Glv Pro-Ala), , (Gly-Pro-MePro), , (Pro-Gly-Pro), , and (Pro-Gly-FPro),Pro, were tested for inhibition of hydroxylation over the range n = 1 to n = 4. Inhibition is first observed at n = 2 and for these short oligopeptides is maximal between n = 3 and n = 4. Secondary structure appears to be less important than primary structure in determining whether a peptide will be an inhibitor of the hydroxylase. It is suggested that in a polypeptide chain a specific sequence of 6-9 amino acids is required to identify an appropriate prolyl residue for hydroxylation. Among a series of proline derivatives tested, only 4.fluoro, 3- and 4.methylproline inhibited the hydroxylase.
In animal tissues hydroxyproline is synthesized by the enzymic hydroxylation of selected proline residues in peptide linkage to form hydroxyproline residues. The peptidyl proline hydroxylase has been partially characterized and is a mixed function oxidase requiring molecular oxygen, ascorbic acid, ferrous ion, and cr-ketoglutarate for activity (1, 2). Since collagen is the only animal protein in which hydroxyproline occurs, a major problem in understanding the conversion of bound proline to hydroxyproline concerns the mechanisms whereby the hydroxylating enzyme recognizes the appropriate proline residues to be hydroxylated. It appears likely that those residues selected for hydroxylation are recognized from their position in specific sequences of amino acids. However, it is not known whether the secondary or tertiary structure of the polypeptide strands of collagen influence t)he selection process.
Collagen has a molecular weight of 300,000 and is composed of three separate polypeptide chains of approximately equal size. The individual chains are in the polyproline II conformation and form a triple helix (3). A glycine residue is present in every third position throughout most of their amino acid sequence. Polytripeptides such as H(Pro-Gly-Pro),OH (4), H(Gly-Pro-Gly),OH, and H(Gly-Pro-Ala),OH (5) have been synthesized and tested as model substrate or inhibitors of the hydroxylating enzyme? The polytripeptide H(Pro-Gly-Pro),OH can serve as substrate of peptidyl proline hydroxylase (6). Over certain ranges of molec1 Incubation of a variety of tissues with labeled proline under conditions where hydroxylation is inhibited yields proline-labeled material with properties of collagen but with no labeled hyd roxyproline. The term “protocollagen” has been given t,o this experimentally produced material by D. J. Prockop and colleagues (3). 779
HUTTON,
ular weight, large polymers of repeating tripeptide units have greater affinities for the enzyme than polymers of identical repeating unit but lower molecular weight (6). Poly-L-proline II is a competitive inhibitor of the hydroxylating reaction (6), whereas free proline, hydroxyproline, and several tripeptides containing proline have no effect on the reaction (7). The purpose of this investigation was to define more clearly the affinity of collagen proline hydroxylase for various polypeptides. A number of synthetic polymers differing in both sequenceand molecular weight have been synthesized. Whether these synthetic oligo- and polypeptides can serve as substrates and inhibitors of collagen proline hydroxylase has been tested in a cell-free hydroxylating system. MATERIALS Small quantities of of proline were kindly
AND METHODS the following supplied by
derivatives Drs. A.
B.
Mauger, Washington Hospital Center, Washington, D.C.; and J. Kollonitsch, Merck Sharp and Dohme, Rahway, New Jersey: cis- and trans4fluoro; cis- and trans4-chloro; cis- and translbromo; 4-methyl, mixed isomers; and cis- and trams-3-methyl proline. Each had been checked for authenticity during the course of studies on the chemistry of proline and its derivatives (8). Na-Boc-amino acids2 were either synthesized or purchased from Cycle Chemical Corporation, Los Angeles. Unless otherwise specified, oligopeptides of defined amino acid sequence and length were synthesized by the solid phase method, using the modifications of Stewart and Woolley (9, 10). After cleavage from the resin, peptides were homogeneous on thin-layer chromatography and gave correct quantitative amino acid analyses. The oligopeptides H(Gly-Pro-Ala)OH, H(GlyPro-Ala)zOH, H(Gly-Pro-Ala)40H, and H(GlyPro-Ala),OH, mol. wt. 14,OQO, were kindly supplied by Dr. E. R. Blout, Harvard University (5). 3,4-3H-Proline, 5.0 C/mmole, was purchased from New England Nuclear Corporation, Boston, Massachusetts. Two batches of ([3,4-SH-Pro]Gly-Pro), with specific activities of 1.0 &/mg and 2.1 rC/mg were prepared from benzyloxycarbonyl-L-3 ,4-3H-prolyl chloride and nonlabeled H(Gly-Pro)OH HBr according to methods previously reported (4). The average molecular 2 Abbreviations used: Na-Boc, N-tertiary oxycarbonyl; MePro, trans-3-methylproline; trans-4-fluoroproline.
butylFPro,
ET AL. TABLE INHIBITION DUES
ACID
OF PROLYL ANALOGUES
RESI-
PROLINE
OF
Proline
I
OF HYDROXYLATION BY FREE IMINO
derivative”
Proline
Proline trans-4-hydroxy cis-4-fluoro trans-4-fluoro
0 0 30 30
cis-4-chloro trans-4-chloro
0 0
Inhibition (%sJ
derivative”
cisl-bromo trans-4-bromo 4-methyl (mixed isomer) cis-3-methyl trams-3-methyl
1
0 0 30 30 30
a All compounds were tested at a final concentration of 0.001 M in the standard complete hydroxylatingsystem using 3 ,4-3H-labeled, hydroxyproline-deficient protein &s substrate. Each tube contained 2.7 mg chick AS60 enzyme/ml. Incubations were at 30” for 30 minutes. b No inhibition corresponded to the formation of 930 cpm of tritiated water with a blank of 30 cpm. Zero inhibition includes values between 0 and 10%. Values represent the average of at least two determinations and are rounded off to the nearest ten. weight of the first batch was 2700; that of the second was 3786 as determined by ultracentrifugation in water. After synthesis the radioactive polymer was chromatographed on a column of Sephadex G-50 (Pharmacia), fine bead form, and the molecular weights of the various fractions were estimated by ultracentrifugation or measurement of specific optical rotation (4), or both. Amino acid content was checked by quantitative amino acid analysis. Nonradioactive poly-L-proline II, mol. wt. 9@30, and (Pro-Gly-Pro), , mol. wt. 4358, used in certain experiments, have been described previously (4). Gelatinized, hydroxyproline-deficient protein that serves as substrate for collagen proline hydroxylase was prepared by incubating minces of chick embryo with 3,4-*H-proline in the presence of ol,&dipyridyl as previously described (11). Collagen proline hydroxylase was prepared from 105,000 g supernatant fractions obtained from homogenates of 7- to S-day-old chick embryos (1). The supernatant enzyme was purified either by precipitation with ammonium sulfate to yield a 60% ammonium sulfate fraction (AS60 enzyme) (1) or by chromatography on DEAE-Sephadex to yield 5- to lo-fold purified DEAE enzyme.3 To 3 Rhoads, unpublished
R., Hutton, procedure.
J.,
and
Udenfriend,
S.,
COLLAGEN
PROLINE
assay the conversion of proline to hydroxyproline, the standard complete hydroxylating system (1) contained (in micromoles): Tris-HCl, pH 7.5, 200; a-ketoglutarate, 0.2; ascorbate, 1.0; ferrous ion as Fe(NH,)2(S01)2, 0.2; peptidyl proline approximately 809,090 dpm or 3Hsubstrate, labeled polymer as specified in the text; enzyme as specified; and water to 2 ml. The mixtures were incubated at 30” for 20 minutes in air with shaking unless otherwise specified. Reactions were stopped by adding 0.1 volume of 50% trichloroacetic acid. Tritiated water was separated from the reaction mixtures by vacuum distillation, and aliquom were counted in a Packard Tri-Carb scintillation spectrometer (11). Radioactivity is reported as counts per minute (cpm) and is corrected to 10% counting efficiency. The amount of tritiated water formed with synthetic substrate was proportional to the amount of hydroxyproline produced by the procedures described previously (II). RESULTS
Proline derivatives as inhibitors. A number of analogues of proline were tested for their ability to inhibit the hydroxylation of peptidyl-proline substrate (Table I). 4Fluoro, 3-methyl, and 4-methyl derivatives of proline were moderately inhibitory, whereas other derivatives tested were totally without effect at 0.001 M. Because of the small degree of inhibition observed and the small quantities of derivatives available, the mode of action of the inhibitory compounds was not investigated further. Oligo- and polypeptides as inhibitors. Since hydroxylation was not inhibited by high concentrations of proline, hydroxyproline, Gly-Pro-Pro, and Gly-Pro-Hyp, a series of higher molecular weight polymers was testfed. Within the limits of experimental error, poly-L-proline II and H(Pro-GlyPro),OH were competitive inhibitors of the hydroxylation reaction (Fig. l), whereas poly-L-hydroxyproline, dextran, and polyvinylpyrrolidone were not inhibitory at similar concentrations. After these observations, several series of oligopeptides of increasing molecular weights were synthesized and tested for their ability to inhibit the hydroxylation of the 3, 4-3H-proline labeled substrate obtained from chick embryos (Table II). In all series tested the t#ripeptide was either not inhibitory or very
781
HYDROXYLASE
C
IO f
20
id
FIG. 1. Poly-n-proline II and (Pro-Gly-Pro), inhibition of the hydroxylation of substrate prepared from chick embryo. Standard hydroxylating system containing 0.4 mg purified DEAE enzyme per assay. The maximal water release from 0.5 ml of substrate was 3090 cpm. Substrate concentration was measured as milliliters added to the assay; velocity was measured as counts/minute tritiated water per 20 minutes. Final concentrations and molecular weights of inhibiting polymers: poly-n-proline II, mol. wt. 9000, 0.1 mg/ml, 0 ; (Pro-Gly-Pro), , mol. wt. 4353, 0.3 mg/ml, 0; no inhibitor added, 0.
slightly so. Nonapeptides and dodecapeptides were inhibitory; hexapeptides were less inhibit’ory than the larger peptides. In the case of H(Gly-Pro-Ala),OH, a higher molecular weight (14,000) polymer was tested, and in this case at equal weight concentrations, the dodecapeptide appeared to be as effective an inhibitor as the polymer. Polymers of Gly-Pro-Ala do not show regular structure in solution (5), whereas polymers of Pro-Gly-Pro do show helical conformation in solution and an increase in the extent of helicity with an increase in chain length (4). Since oligopeptides of both repeating tripeptide units are equally inhibit,ory, inhibition by the peptides tested would appear to be related to sequenceand length rather than to conformation. Inhibition of substrate hydroxylation by oligopeptides was reversed by increasing the concentration of the protein substrate in a manner similar to that shown for polymers
782
HUTTON, TABLE INHIBITION
OF
HYDROXYLATION
ET II
OF PROLINE
Inhibition
(%)b
AL.
RESIDUES Oligopeptid.@
Gly-Pro-Ala (Gly-Pro-Ala)2 (Gly-Pro-Ala)4 (Gly-Pro-Ala).
0 30 70 50
Pro-Gly-Pro (Pro-Gly-Pro)2 (Pro-Gly-Pro)3 (Pro-Gly-Pro)
Gly-Pro-MePro (Gly-Pro-MePro)* (Gly-Pro-MePro) (Gly-Pro-MePro)a
20 30 70 70
(Pro-Gly-FPro)Pro (Pro-Gly-FPro)pPro (Pro-Gly-FPro)3Pro (Pro-Gly-FPro)dPro
3
BY
OLIGOPEPTIDES Inhibition
(%)b
0 30 50 70
4 Not
available 20 30 30
a All peptides were tested at a final concentration of 0.5 mg/ml in the standard complete hydroxylating system containing 3,4-*H-proline-labeled, hydroxyproline-deficient protein as substrate and 0.2 mg purified DEAE enzyme/ml. Incubation was at 30” for 20 minutes. (Gly-Pro-Ala), was of 14,000 molecular weight (5). b As in Table I.
2.0 c
*
MW ,300
I I
10
1 20
$ (mghlj’
FIG. 2. Double reciprocal plots of l/V versus l/S. Velocity is expressed as millimicromoles hydroxyproline formed per minute for hydroxylation of four different weight fractions of ([3,4-3HPro]-Gly-Pro), , batch 1. Substrate concentration is expressed as milligrams polymer per milliliter incubation. Standard hydroxylating system contained 7 mg/ml AS60 enzyme; incubation time was 20 minutes. One mpmole hydroxyproline is equivalent to 24 cpm tritiated water. Reagent blanks including background were 30 cpm or less.
in Fig. 1, indicating that the inhibitory oligopeptides and polypeptides are probably competitive inhibitors of the enzyme. Oligopeptides containing trans-3-methylproline and transl-fluoroproline were synthesized since these free imino acids had inhibited the hydroxylase (Table I). However, the
resulting oligopeptides were no more inhibitory than oligopeptides containing natural proline itself. Polytripeptides as substrates. Fractions of H([3,4-3H-Pro]-Gly-Pro),0H of various average molecular weights were prepared and utilized as substrate for the hydroxylase. For batch 1 of the polymer, 24 cpm tritiated water (10 % efficiency of counting) were released per millimicromole of hydroxyproline formed; for batch 2 of the polymer 58 cpm tritiated water were released per millimicromole of hydroxyproline. With the standard complete hydroxylating system, the rate of polymer hydroxylation was linear for 3540 minutes and then decreased. In kinetic experiments incubations were for 20 minutes. The amount of tritiated water was assayed at this time and used as a measure of initial velocity. Figure 2 shows double reciprocal plots of l/ii versus l/S for hydroxylation of four different weight fractions of H([3,4-3H-Pro]-Gly-Pro),0H, batch 1, with AS60 enzyme (7 mg/ml). Similar plots were obtained with purified DEAE enzyme (1 mg/ml) and fractions of batch 2 of the polymer. Apparent K, values for fractions of H([3 ,4-3H-Pro]Gly-Pro),OH of different average molecular weights were calculated from these plots (Table III). In all cases the slopes of the double reciprocal plots increased with decreasing substrate concentrations so that
COLLA(:EN TABLE
PROLINE
783
HYDROXYLASE
III
K, VALUES FOH FIL~CTIONS OF ([3,43H-P~~]-G~~-P~~), OF INCREASING AVERAGE MOLECULAR WEIGHTS
APPARENT
Polymer batch”
1
2
Average mol. wt.
Apparent mg /ml
Km IDM
1300 2200 2700 4000
4.0 0.53 0.25
3.1 0.50 0.20 0.062
2400 4100 8ooo
0.41 0.14 0.042
0.17 0.034 0.0053
1.1
1~Polymer fractions were tested in the standard hydroxylating system, and apparent K, values were estimated from double reciprocal plots (e.g., Fig. 2). For batch 1, condit,ions are described in the legend to Fig. 2, and average molecular weights were determined by ultracentrifugation. For batch 2, 2 mg of purified DEAE enzyme was used per assay tube. In this case average molecular weights were estimated both from measurements of specific optical rotation and by ultracentrifugation to determine the weight average of molecular weight for the batch followed by chromatography on calibrated columus of Sephadex G-50.
the intercept on the abscissawas estimated by extrapolation (Fig. 2). Apparent K, values calculated from data obtained in experiments conducted nearly 1 year apart with two different batches of polymer and two different methods of enzyme preparation were in good agreement. A plot on a semi-log scaleof apparent K, versus average molecular weight (Fig. 3) shows that the affinity of the enzyme for higher molecular weight substrates is much greater than for smaller substrates. From the data it would appear that maximal affinity would be exhibited by a polymer of molecular weight considerably greater than 8000. Although the apparent K, decreased as molecular weight increased, the maximal different velocity of reactions utilizing weight fractions of polymer appeared to be identical. This is in agreement with observations of Kivirikko and Prockop (6). The Tim,, estimated from Fig. 2 was 10 mpmoles of hydroxyproline per minute for all weight fractions of polymer using AS60
0.001
’ 0
’ 2,000 AVERAGE
I 1 4,000 6,000 MOLECULAR
I 8,000 WEIGHT
I 10,000
FIG. 3. Plot of apparent K, as a function of weight-average molecular weight of fractions of ([3,4-3H-Pro)-Gly-Pro), . The K, is plotted on a log scale; molecular weight on a linear scale. Circles indicate K, in mM; squares indicate K, in mg/ml; open symbols represent those obtained using batch 1 of polymer plus AS60 enzyme; filled symbols represent those obtained using batch 2 of polymer plus DEAE enzyme.
enzyme. This corresponds to a V,, of 5.9 pg of hydroxyproline per milligram of enzyme protein per hour and is approximately half that observed by Kivirikko and Prockop (6) using their enzyme preparation and unlabeled (Pro-Gly-Pro), . In the present studies the specific activity of purified DEAE enzyme is approximately five times as great as that of AS60 enzyme. From these data it appears that the specific activity of preparations of our AS60 enzyme is half as great as that of preparations used by Kivirikko and Prockop (6), but that our DEAE enzyme is somewhat more highly purified than the enzyme which they used. DISCUSSION
Clarification of the mechanisms whereby a proline residue is selected for hydroxylation is in its early stages.With a seriesof fractions of (Pro-Gly-Pro),, of varying average molecular weights, it has been shown that over certain ranges the affinity of collagen
784
HUTTON,
proline hydroxylase is greater for high molecular weight polypeptides than for those of low molecular weight. Since fractions of randomly polymerized polytripeptides obtained by gel filtration are not of uniform molecular weight, it is possible that all the observed activity with the smallest oligopeptides was due to the presence of small amounts of larger polymers having a molecular weight greater than some critical value such as 10,000. However, such a possibility has been ruled out by the unequivocal synthesis of the dodecapeptide (Gly-Pro[3, 4-3H-Pro])3-Gly-Pro-Pro (mol. wt. 1104), which has been shown to serve as substrate for collagen proline hydroxylase4 as predicted by Fig. 3. From Fig. 3 one would also predict that maximal affinity for the enzyme will be exhibited by polypeptides of molecular weight greater than 8000. With 14C-labeled polypeptides, Prockop et al. (12) concluded that the ability of (Pro-Gly-Pro), to serve as substrate was proportional to the average molecular weight of different polymer fractions over the weight range tested. However, in a later publication, Kivirikko and Prockop (6) concluded that the apparent K, remained constant for molecular weights over 4000 and that the affinity for polypeptide did not appear to be proportional to molecular weight of substrate. The reasons for the discrepancy between the two reports are not clear. The V,,, was not affected by changes in the molecular weight of substrate over the weight range tested. During the course of a 20-minute incubation no more than 5-10% of the labeled proline residues in ([3 ,4-3H-Pro]-Gly-Pro), are hydroxylated. For example, 8 % of the labeled proline residues of a polymer of 4000 average molecular weight (0.1 mg/ml) were hydroxylated in 20 minutes and released 1370 cpm of tritiated water (Fig. 2). When the incubation time was increased, kinetics became nonlinear. It was therefore not possible to use polymers of identical sequence but lower specific activity simply by increasing the time of incubation. 4 Marglin, A., Rhoads, published observations.
and
Udenfriend,
S., un-
ET
AL.
Polymers of two specific activities, 1.0 and 2.1 $Z/mg, have been synthesized. Both were unstable due to cumulative radiation damage and could not serve as substrate after about 6 weeks of storage at -15” whether in aqueous solution or dry. Unless better storage conditions are found it may not be feasible to use these polymers as substrates for routine purposes.5 In lieu of finding better storage conditions, an approach to the problem of preparing stable tritiated substrate might be the insertion of residues other than proline in order to gain adequate molecular weight for achain containing a limited number of hydroxylatable labeled sequences such as Gly-X-(3 ,4-3H-Pro). In this way a high affinity polymer might be made in which specific activity as &/pmole proline would be high, but as rC/mg polymer would be lower than 1.0. It is also possible that a polymer of different sequence would have a greater percentage of hydroxylatable proline residues. That this approach is feasible is suggested by the fact that tritium-labeled substrate prepared from natural sources (11) has high affinity for the enzyme and is stable for several months at -15”. A number of derivatives of free proline was tested for their ability to inhibit hydroxylation. The 3- and 4-methyl and 4fluoro derivatives were weakly inhibitory. The nature and significance of the inhibition remains to be evaluated. When several series of oligopeptides were tested, none of the tripeptides was significantly inhibitory, whereas, hexa-, nona-, and dodecapeptides were effective. It was initially felt that the smallest inhibitory peptide (hexapeptide) might also be the smallest peptide capable of possessing secondary structure. However, oligopeptides of the (Gly-ProAla), series behave like members of the series even though the (Pro-Gly-Pro), first exist as a random coil in solution (5), 6 In more recent studies R. Rhoads has shown that the tritium labeled synthetic peptides do not deteriorate when dissolved in water containing 2% ethanol and stored at the temperature of liquid nitrogen.
COLLAGEN
PROLINE
whereas the second probably possesses a certain degree of ordered conformation (4). Competition between polypeptide substrate and oligopeptide could result either from combination of the oligopeptide with the active hydroxylating site on the enzyme or from combination of oligopeptide with polypeptide substrate. If the first explanation is correct, then the results suggest that it requires a sequence of 6-9 amino acids to make a specific proline residue eligible for hydroxylation. Since the sequences of most of the oligopeptides and polypeptides used in the present study resemble each other, no conclusions can be drawn as to what sequences make specific proline residues eligible for hydroxylation. The synthesis of a greater variety of proline-contaming polypeptides and a better knowledge of the primary sequence of collagen are prerequisites for a better understanding of this challenging problem.
785
HYDROXYLASE REFERENCES
1. HUTTON, J. J., TAPPEL, A. L., AND UDENFRIEND, S., Arch. Biochem. Biophys. 118,231 (1967). 2. KIVIRIKKO, K. I., AND PROCKOP, D. J., Proc Natl. Acad. Sci. U.S. 67, 782 (1967). 3. HARRINGTON, W. F., AND VON HIPPEL, P. H., Advan. Protein Chem. 18, 1 (1961). 4. ENGEL, J., KURTZ, J., KATCHALSKI, E., AND BERGER, A., J. Mol. Biol. 1’7, 255 (1966). 5. ORIEL, P. J., AND BLOUT, E. R., J. Am. Chem. Sot. 88, 2041 (1966). 6. KIVIRIKKO, K. I., AND PROCKOP, D. J., J. Biol. Chem. 342, 4007 (1967). 7. HUTTON, J. J., AND UDENFRIEND, S., Proc. Natl. Acad. Sci. U.S. 68, 198 (1966). 8. MAUGER, A. B., AND WITKOP, B., Chem. Rev. 66, 47 (1966). 9. MERRIFIELD, R. B., Science 160, 178 (1965). 10. STEWART, J. M., AND WOOLLEY, D. W., iVature 206, 619 (1965). 11. HUTTON, J. J., TAPPEL, A. L., AND UDENFRIEND, S., Anal. Biochem. 16,3&4 (1966). 12. PROCKOP, D. J., JUVA, K., AND ENGEL, J., Z. Physiol. Chem. 348, 553 (1967).
OF
BIOCHEMISTRY
Synthetic
AND
BIOPHYSICS
Polypeptides
779-785
(1968)
as Substrates Proline
J. J. HUTTON,
126,
JR., A. MARGLIN,
and
Inhibitors
of Collagen
Hydroxylase B. WITKOP,
J. KURT&
A. BERGER,
AND
S. UDENFRlEND Laboratory Inslitute
of Clinical of Arthritis Department
Biochemistry, and Metabolic of Biophysics,
National Heart Institute, and Laboratory of Chemistry, National Diseases, National Institutes of Health, Bethesda, Maryland; The Weizmann Institute of Science, Rehoooth, Israel Received
February
1, 1968
The synthetic polypeptide ([3,4-3H-Pro]-Gly-Pro), can be hydroxylated by collagen proline hydroxylase. The affinity of the enzyme for the substrate increases with increasing molecular weight of substrate over the range 1300-8000. The data indicate that maximal affinity should be observed with a polypeptide of molecular weight considerably greater than 8000. Poly-L-proline II and (Pro-Gly-Pro), appear to be COIV petitive inhibitors of the hydroxylation reaction. The series of oligopeptides, (Glv Pro-Ala), , (Gly-Pro-MePro), , (Pro-Gly-Pro), , and (Pro-Gly-FPro),Pro, were tested for inhibition of hydroxylation over the range n = 1 to n = 4. Inhibition is first observed at n = 2 and for these short oligopeptides is maximal between n = 3 and n = 4. Secondary structure appears to be less important than primary structure in determining whether a peptide will be an inhibitor of the hydroxylase. It is suggested that in a polypeptide chain a specific sequence of 6-9 amino acids is required to identify an appropriate prolyl residue for hydroxylation. Among a series of proline derivatives tested, only 4.fluoro, 3- and 4.methylproline inhibited the hydroxylase.
In animal tissues hydroxyproline is synthesized by the enzymic hydroxylation of selected proline residues in peptide linkage to form hydroxyproline residues. The peptidyl proline hydroxylase has been partially characterized and is a mixed function oxidase requiring molecular oxygen, ascorbic acid, ferrous ion, and cr-ketoglutarate for activity (1, 2). Since collagen is the only animal protein in which hydroxyproline occurs, a major problem in understanding the conversion of bound proline to hydroxyproline concerns the mechanisms whereby the hydroxylating enzyme recognizes the appropriate proline residues to be hydroxylated. It appears likely that those residues selected for hydroxylation are recognized from their position in specific sequences of amino acids. However, it is not known whether the secondary or tertiary structure of the polypeptide strands of collagen influence t)he selection process.
Collagen has a molecular weight of 300,000 and is composed of three separate polypeptide chains of approximately equal size. The individual chains are in the polyproline II conformation and form a triple helix (3). A glycine residue is present in every third position throughout most of their amino acid sequence. Polytripeptides such as H(Pro-Gly-Pro),OH (4), H(Gly-Pro-Gly),OH, and H(Gly-Pro-Ala),OH (5) have been synthesized and tested as model substrate or inhibitors of the hydroxylating enzyme? The polytripeptide H(Pro-Gly-Pro),OH can serve as substrate of peptidyl proline hydroxylase (6). Over certain ranges of molec1 Incubation of a variety of tissues with labeled proline under conditions where hydroxylation is inhibited yields proline-labeled material with properties of collagen but with no labeled hyd roxyproline. The term “protocollagen” has been given t,o this experimentally produced material by D. J. Prockop and colleagues (3). 779
HUTTON,
ular weight, large polymers of repeating tripeptide units have greater affinities for the enzyme than polymers of identical repeating unit but lower molecular weight (6). Poly-L-proline II is a competitive inhibitor of the hydroxylating reaction (6), whereas free proline, hydroxyproline, and several tripeptides containing proline have no effect on the reaction (7). The purpose of this investigation was to define more clearly the affinity of collagen proline hydroxylase for various polypeptides. A number of synthetic polymers differing in both sequenceand molecular weight have been synthesized. Whether these synthetic oligo- and polypeptides can serve as substrates and inhibitors of collagen proline hydroxylase has been tested in a cell-free hydroxylating system. MATERIALS Small quantities of of proline were kindly
AND METHODS the following supplied by
derivatives Drs. A.
B.
Mauger, Washington Hospital Center, Washington, D.C.; and J. Kollonitsch, Merck Sharp and Dohme, Rahway, New Jersey: cis- and trans4fluoro; cis- and trans4-chloro; cis- and translbromo; 4-methyl, mixed isomers; and cis- and trams-3-methyl proline. Each had been checked for authenticity during the course of studies on the chemistry of proline and its derivatives (8). Na-Boc-amino acids2 were either synthesized or purchased from Cycle Chemical Corporation, Los Angeles. Unless otherwise specified, oligopeptides of defined amino acid sequence and length were synthesized by the solid phase method, using the modifications of Stewart and Woolley (9, 10). After cleavage from the resin, peptides were homogeneous on thin-layer chromatography and gave correct quantitative amino acid analyses. The oligopeptides H(Gly-Pro-Ala)OH, H(GlyPro-Ala)zOH, H(Gly-Pro-Ala)40H, and H(GlyPro-Ala),OH, mol. wt. 14,OQO, were kindly supplied by Dr. E. R. Blout, Harvard University (5). 3,4-3H-Proline, 5.0 C/mmole, was purchased from New England Nuclear Corporation, Boston, Massachusetts. Two batches of ([3,4-SH-Pro]Gly-Pro), with specific activities of 1.0 &/mg and 2.1 rC/mg were prepared from benzyloxycarbonyl-L-3 ,4-3H-prolyl chloride and nonlabeled H(Gly-Pro)OH HBr according to methods previously reported (4). The average molecular 2 Abbreviations used: Na-Boc, N-tertiary oxycarbonyl; MePro, trans-3-methylproline; trans-4-fluoroproline.
butylFPro,
ET AL. TABLE INHIBITION DUES
ACID
OF PROLYL ANALOGUES
RESI-
PROLINE
OF
Proline
I
OF HYDROXYLATION BY FREE IMINO
derivative”
Proline
Proline trans-4-hydroxy cis-4-fluoro trans-4-fluoro
0 0 30 30
cis-4-chloro trans-4-chloro
0 0
Inhibition (%sJ
derivative”
cisl-bromo trans-4-bromo 4-methyl (mixed isomer) cis-3-methyl trams-3-methyl
1
0 0 30 30 30
a All compounds were tested at a final concentration of 0.001 M in the standard complete hydroxylatingsystem using 3 ,4-3H-labeled, hydroxyproline-deficient protein &s substrate. Each tube contained 2.7 mg chick AS60 enzyme/ml. Incubations were at 30” for 30 minutes. b No inhibition corresponded to the formation of 930 cpm of tritiated water with a blank of 30 cpm. Zero inhibition includes values between 0 and 10%. Values represent the average of at least two determinations and are rounded off to the nearest ten. weight of the first batch was 2700; that of the second was 3786 as determined by ultracentrifugation in water. After synthesis the radioactive polymer was chromatographed on a column of Sephadex G-50 (Pharmacia), fine bead form, and the molecular weights of the various fractions were estimated by ultracentrifugation or measurement of specific optical rotation (4), or both. Amino acid content was checked by quantitative amino acid analysis. Nonradioactive poly-L-proline II, mol. wt. 9@30, and (Pro-Gly-Pro), , mol. wt. 4358, used in certain experiments, have been described previously (4). Gelatinized, hydroxyproline-deficient protein that serves as substrate for collagen proline hydroxylase was prepared by incubating minces of chick embryo with 3,4-*H-proline in the presence of ol,&dipyridyl as previously described (11). Collagen proline hydroxylase was prepared from 105,000 g supernatant fractions obtained from homogenates of 7- to S-day-old chick embryos (1). The supernatant enzyme was purified either by precipitation with ammonium sulfate to yield a 60% ammonium sulfate fraction (AS60 enzyme) (1) or by chromatography on DEAE-Sephadex to yield 5- to lo-fold purified DEAE enzyme.3 To 3 Rhoads, unpublished
R., Hutton, procedure.
J.,
and
Udenfriend,
S.,
COLLAGEN
PROLINE
assay the conversion of proline to hydroxyproline, the standard complete hydroxylating system (1) contained (in micromoles): Tris-HCl, pH 7.5, 200; a-ketoglutarate, 0.2; ascorbate, 1.0; ferrous ion as Fe(NH,)2(S01)2, 0.2; peptidyl proline approximately 809,090 dpm or 3Hsubstrate, labeled polymer as specified in the text; enzyme as specified; and water to 2 ml. The mixtures were incubated at 30” for 20 minutes in air with shaking unless otherwise specified. Reactions were stopped by adding 0.1 volume of 50% trichloroacetic acid. Tritiated water was separated from the reaction mixtures by vacuum distillation, and aliquom were counted in a Packard Tri-Carb scintillation spectrometer (11). Radioactivity is reported as counts per minute (cpm) and is corrected to 10% counting efficiency. The amount of tritiated water formed with synthetic substrate was proportional to the amount of hydroxyproline produced by the procedures described previously (II). RESULTS
Proline derivatives as inhibitors. A number of analogues of proline were tested for their ability to inhibit the hydroxylation of peptidyl-proline substrate (Table I). 4Fluoro, 3-methyl, and 4-methyl derivatives of proline were moderately inhibitory, whereas other derivatives tested were totally without effect at 0.001 M. Because of the small degree of inhibition observed and the small quantities of derivatives available, the mode of action of the inhibitory compounds was not investigated further. Oligo- and polypeptides as inhibitors. Since hydroxylation was not inhibited by high concentrations of proline, hydroxyproline, Gly-Pro-Pro, and Gly-Pro-Hyp, a series of higher molecular weight polymers was testfed. Within the limits of experimental error, poly-L-proline II and H(Pro-GlyPro),OH were competitive inhibitors of the hydroxylation reaction (Fig. l), whereas poly-L-hydroxyproline, dextran, and polyvinylpyrrolidone were not inhibitory at similar concentrations. After these observations, several series of oligopeptides of increasing molecular weights were synthesized and tested for their ability to inhibit the hydroxylation of the 3, 4-3H-proline labeled substrate obtained from chick embryos (Table II). In all series tested the t#ripeptide was either not inhibitory or very
781
HYDROXYLASE
C
IO f
20
id
FIG. 1. Poly-n-proline II and (Pro-Gly-Pro), inhibition of the hydroxylation of substrate prepared from chick embryo. Standard hydroxylating system containing 0.4 mg purified DEAE enzyme per assay. The maximal water release from 0.5 ml of substrate was 3090 cpm. Substrate concentration was measured as milliliters added to the assay; velocity was measured as counts/minute tritiated water per 20 minutes. Final concentrations and molecular weights of inhibiting polymers: poly-n-proline II, mol. wt. 9000, 0.1 mg/ml, 0 ; (Pro-Gly-Pro), , mol. wt. 4353, 0.3 mg/ml, 0; no inhibitor added, 0.
slightly so. Nonapeptides and dodecapeptides were inhibitory; hexapeptides were less inhibit’ory than the larger peptides. In the case of H(Gly-Pro-Ala),OH, a higher molecular weight (14,000) polymer was tested, and in this case at equal weight concentrations, the dodecapeptide appeared to be as effective an inhibitor as the polymer. Polymers of Gly-Pro-Ala do not show regular structure in solution (5), whereas polymers of Pro-Gly-Pro do show helical conformation in solution and an increase in the extent of helicity with an increase in chain length (4). Since oligopeptides of both repeating tripeptide units are equally inhibit,ory, inhibition by the peptides tested would appear to be related to sequenceand length rather than to conformation. Inhibition of substrate hydroxylation by oligopeptides was reversed by increasing the concentration of the protein substrate in a manner similar to that shown for polymers
782
HUTTON, TABLE INHIBITION
OF
HYDROXYLATION
ET II
OF PROLINE
Inhibition
(%)b
AL.
RESIDUES Oligopeptid.@
Gly-Pro-Ala (Gly-Pro-Ala)2 (Gly-Pro-Ala)4 (Gly-Pro-Ala).
0 30 70 50
Pro-Gly-Pro (Pro-Gly-Pro)2 (Pro-Gly-Pro)3 (Pro-Gly-Pro)
Gly-Pro-MePro (Gly-Pro-MePro)* (Gly-Pro-MePro) (Gly-Pro-MePro)a
20 30 70 70
(Pro-Gly-FPro)Pro (Pro-Gly-FPro)pPro (Pro-Gly-FPro)3Pro (Pro-Gly-FPro)dPro
3
BY
OLIGOPEPTIDES Inhibition
(%)b
0 30 50 70
4 Not
available 20 30 30
a All peptides were tested at a final concentration of 0.5 mg/ml in the standard complete hydroxylating system containing 3,4-*H-proline-labeled, hydroxyproline-deficient protein as substrate and 0.2 mg purified DEAE enzyme/ml. Incubation was at 30” for 20 minutes. (Gly-Pro-Ala), was of 14,000 molecular weight (5). b As in Table I.
2.0 c
*
MW ,300
I I
10
1 20
$ (mghlj’
FIG. 2. Double reciprocal plots of l/V versus l/S. Velocity is expressed as millimicromoles hydroxyproline formed per minute for hydroxylation of four different weight fractions of ([3,4-3HPro]-Gly-Pro), , batch 1. Substrate concentration is expressed as milligrams polymer per milliliter incubation. Standard hydroxylating system contained 7 mg/ml AS60 enzyme; incubation time was 20 minutes. One mpmole hydroxyproline is equivalent to 24 cpm tritiated water. Reagent blanks including background were 30 cpm or less.
in Fig. 1, indicating that the inhibitory oligopeptides and polypeptides are probably competitive inhibitors of the enzyme. Oligopeptides containing trans-3-methylproline and transl-fluoroproline were synthesized since these free imino acids had inhibited the hydroxylase (Table I). However, the
resulting oligopeptides were no more inhibitory than oligopeptides containing natural proline itself. Polytripeptides as substrates. Fractions of H([3,4-3H-Pro]-Gly-Pro),0H of various average molecular weights were prepared and utilized as substrate for the hydroxylase. For batch 1 of the polymer, 24 cpm tritiated water (10 % efficiency of counting) were released per millimicromole of hydroxyproline formed; for batch 2 of the polymer 58 cpm tritiated water were released per millimicromole of hydroxyproline. With the standard complete hydroxylating system, the rate of polymer hydroxylation was linear for 3540 minutes and then decreased. In kinetic experiments incubations were for 20 minutes. The amount of tritiated water was assayed at this time and used as a measure of initial velocity. Figure 2 shows double reciprocal plots of l/ii versus l/S for hydroxylation of four different weight fractions of H([3,4-3H-Pro]-Gly-Pro),0H, batch 1, with AS60 enzyme (7 mg/ml). Similar plots were obtained with purified DEAE enzyme (1 mg/ml) and fractions of batch 2 of the polymer. Apparent K, values for fractions of H([3 ,4-3H-Pro]Gly-Pro),OH of different average molecular weights were calculated from these plots (Table III). In all cases the slopes of the double reciprocal plots increased with decreasing substrate concentrations so that
COLLA(:EN TABLE
PROLINE
783
HYDROXYLASE
III
K, VALUES FOH FIL~CTIONS OF ([3,43H-P~~]-G~~-P~~), OF INCREASING AVERAGE MOLECULAR WEIGHTS
APPARENT
Polymer batch”
1
2
Average mol. wt.
Apparent mg /ml
Km IDM
1300 2200 2700 4000
4.0 0.53 0.25
3.1 0.50 0.20 0.062
2400 4100 8ooo
0.41 0.14 0.042
0.17 0.034 0.0053
1.1
1~Polymer fractions were tested in the standard hydroxylating system, and apparent K, values were estimated from double reciprocal plots (e.g., Fig. 2). For batch 1, condit,ions are described in the legend to Fig. 2, and average molecular weights were determined by ultracentrifugation. For batch 2, 2 mg of purified DEAE enzyme was used per assay tube. In this case average molecular weights were estimated both from measurements of specific optical rotation and by ultracentrifugation to determine the weight average of molecular weight for the batch followed by chromatography on calibrated columus of Sephadex G-50.
the intercept on the abscissawas estimated by extrapolation (Fig. 2). Apparent K, values calculated from data obtained in experiments conducted nearly 1 year apart with two different batches of polymer and two different methods of enzyme preparation were in good agreement. A plot on a semi-log scaleof apparent K, versus average molecular weight (Fig. 3) shows that the affinity of the enzyme for higher molecular weight substrates is much greater than for smaller substrates. From the data it would appear that maximal affinity would be exhibited by a polymer of molecular weight considerably greater than 8000. Although the apparent K, decreased as molecular weight increased, the maximal different velocity of reactions utilizing weight fractions of polymer appeared to be identical. This is in agreement with observations of Kivirikko and Prockop (6). The Tim,, estimated from Fig. 2 was 10 mpmoles of hydroxyproline per minute for all weight fractions of polymer using AS60
0.001
’ 0
’ 2,000 AVERAGE
I 1 4,000 6,000 MOLECULAR
I 8,000 WEIGHT
I 10,000
FIG. 3. Plot of apparent K, as a function of weight-average molecular weight of fractions of ([3,4-3H-Pro)-Gly-Pro), . The K, is plotted on a log scale; molecular weight on a linear scale. Circles indicate K, in mM; squares indicate K, in mg/ml; open symbols represent those obtained using batch 1 of polymer plus AS60 enzyme; filled symbols represent those obtained using batch 2 of polymer plus DEAE enzyme.
enzyme. This corresponds to a V,, of 5.9 pg of hydroxyproline per milligram of enzyme protein per hour and is approximately half that observed by Kivirikko and Prockop (6) using their enzyme preparation and unlabeled (Pro-Gly-Pro), . In the present studies the specific activity of purified DEAE enzyme is approximately five times as great as that of AS60 enzyme. From these data it appears that the specific activity of preparations of our AS60 enzyme is half as great as that of preparations used by Kivirikko and Prockop (6), but that our DEAE enzyme is somewhat more highly purified than the enzyme which they used. DISCUSSION
Clarification of the mechanisms whereby a proline residue is selected for hydroxylation is in its early stages.With a seriesof fractions of (Pro-Gly-Pro),, of varying average molecular weights, it has been shown that over certain ranges the affinity of collagen
784
HUTTON,
proline hydroxylase is greater for high molecular weight polypeptides than for those of low molecular weight. Since fractions of randomly polymerized polytripeptides obtained by gel filtration are not of uniform molecular weight, it is possible that all the observed activity with the smallest oligopeptides was due to the presence of small amounts of larger polymers having a molecular weight greater than some critical value such as 10,000. However, such a possibility has been ruled out by the unequivocal synthesis of the dodecapeptide (Gly-Pro[3, 4-3H-Pro])3-Gly-Pro-Pro (mol. wt. 1104), which has been shown to serve as substrate for collagen proline hydroxylase4 as predicted by Fig. 3. From Fig. 3 one would also predict that maximal affinity for the enzyme will be exhibited by polypeptides of molecular weight greater than 8000. With 14C-labeled polypeptides, Prockop et al. (12) concluded that the ability of (Pro-Gly-Pro), to serve as substrate was proportional to the average molecular weight of different polymer fractions over the weight range tested. However, in a later publication, Kivirikko and Prockop (6) concluded that the apparent K, remained constant for molecular weights over 4000 and that the affinity for polypeptide did not appear to be proportional to molecular weight of substrate. The reasons for the discrepancy between the two reports are not clear. The V,,, was not affected by changes in the molecular weight of substrate over the weight range tested. During the course of a 20-minute incubation no more than 5-10% of the labeled proline residues in ([3 ,4-3H-Pro]-Gly-Pro), are hydroxylated. For example, 8 % of the labeled proline residues of a polymer of 4000 average molecular weight (0.1 mg/ml) were hydroxylated in 20 minutes and released 1370 cpm of tritiated water (Fig. 2). When the incubation time was increased, kinetics became nonlinear. It was therefore not possible to use polymers of identical sequence but lower specific activity simply by increasing the time of incubation. 4 Marglin, A., Rhoads, published observations.
and
Udenfriend,
S., un-
ET
AL.
Polymers of two specific activities, 1.0 and 2.1 $Z/mg, have been synthesized. Both were unstable due to cumulative radiation damage and could not serve as substrate after about 6 weeks of storage at -15” whether in aqueous solution or dry. Unless better storage conditions are found it may not be feasible to use these polymers as substrates for routine purposes.5 In lieu of finding better storage conditions, an approach to the problem of preparing stable tritiated substrate might be the insertion of residues other than proline in order to gain adequate molecular weight for achain containing a limited number of hydroxylatable labeled sequences such as Gly-X-(3 ,4-3H-Pro). In this way a high affinity polymer might be made in which specific activity as &/pmole proline would be high, but as rC/mg polymer would be lower than 1.0. It is also possible that a polymer of different sequence would have a greater percentage of hydroxylatable proline residues. That this approach is feasible is suggested by the fact that tritium-labeled substrate prepared from natural sources (11) has high affinity for the enzyme and is stable for several months at -15”. A number of derivatives of free proline was tested for their ability to inhibit hydroxylation. The 3- and 4-methyl and 4fluoro derivatives were weakly inhibitory. The nature and significance of the inhibition remains to be evaluated. When several series of oligopeptides were tested, none of the tripeptides was significantly inhibitory, whereas, hexa-, nona-, and dodecapeptides were effective. It was initially felt that the smallest inhibitory peptide (hexapeptide) might also be the smallest peptide capable of possessing secondary structure. However, oligopeptides of the (Gly-ProAla), series behave like members of the series even though the (Pro-Gly-Pro), first exist as a random coil in solution (5), 6 In more recent studies R. Rhoads has shown that the tritium labeled synthetic peptides do not deteriorate when dissolved in water containing 2% ethanol and stored at the temperature of liquid nitrogen.
COLLAGEN
PROLINE
whereas the second probably possesses a certain degree of ordered conformation (4). Competition between polypeptide substrate and oligopeptide could result either from combination of the oligopeptide with the active hydroxylating site on the enzyme or from combination of oligopeptide with polypeptide substrate. If the first explanation is correct, then the results suggest that it requires a sequence of 6-9 amino acids to make a specific proline residue eligible for hydroxylation. Since the sequences of most of the oligopeptides and polypeptides used in the present study resemble each other, no conclusions can be drawn as to what sequences make specific proline residues eligible for hydroxylation. The synthesis of a greater variety of proline-contaming polypeptides and a better knowledge of the primary sequence of collagen are prerequisites for a better understanding of this challenging problem.
785
HYDROXYLASE REFERENCES
1. HUTTON, J. J., TAPPEL, A. L., AND UDENFRIEND, S., Arch. Biochem. Biophys. 118,231 (1967). 2. KIVIRIKKO, K. I., AND PROCKOP, D. J., Proc Natl. Acad. Sci. U.S. 67, 782 (1967). 3. HARRINGTON, W. F., AND VON HIPPEL, P. H., Advan. Protein Chem. 18, 1 (1961). 4. ENGEL, J., KURTZ, J., KATCHALSKI, E., AND BERGER, A., J. Mol. Biol. 1’7, 255 (1966). 5. ORIEL, P. J., AND BLOUT, E. R., J. Am. Chem. Sot. 88, 2041 (1966). 6. KIVIRIKKO, K. I., AND PROCKOP, D. J., J. Biol. Chem. 342, 4007 (1967). 7. HUTTON, J. J., AND UDENFRIEND, S., Proc. Natl. Acad. Sci. U.S. 68, 198 (1966). 8. MAUGER, A. B., AND WITKOP, B., Chem. Rev. 66, 47 (1966). 9. MERRIFIELD, R. B., Science 160, 178 (1965). 10. STEWART, J. M., AND WOOLLEY, D. W., iVature 206, 619 (1965). 11. HUTTON, J. J., TAPPEL, A. L., AND UDENFRIEND, S., Anal. Biochem. 16,3&4 (1966). 12. PROCKOP, D. J., JUVA, K., AND ENGEL, J., Z. Physiol. Chem. 348, 553 (1967).