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Hydroxylation of (X-Pro-Gly)n by protocollagen proline hydroxylase Effect of chain length, helical conformation and amino acid sequence in the substrate
BIOCHIMICAET BIOPHYSICAACTA
347
BBA 36173 H Y D R O X Y L A T I O N OF (X-PRO-GLY)n BY PROTOCOLLAGEN P R O L I N E HYDROXYLASE E F F E C T OF CHAIN LENGTH, H E L I C A L CONFORMATION AND AMINO ACID SEQUENCE IN T H E SUBSTRATE
KARI I. KIVIRIt(KO*, YASUO KISHIDA, SHUMPEI SAKAKIBARA AND I)AI{XV1N J. PROCKOP** Departments qf .,lledicine and Biochemistry, University of Pennsylvania and the Philadelphia General Hospital, Philadelphia, Pa. 191o4 (U.S.A.) and from the Peptide Center, Institute for Protein Research, Osaka University, Osaka (Japan)
(Received March 3ist, 1972)
SUMMARY
A series of peptides containing sequences o f - X - P r o - G l y - were synthesized by a solid-state method which gave homogeneous products. These peptides were then used to study the synthesis of hydroxyproline by protocollagen proline hydroxylase. Hydroxylation of random-coil forms of the series of peptides (Pro Pro-Gly)n with n = 5, IO, 15 and 20 indicated no difference among the peptides in V but the A'm decreased markedly with chain-length. Hydroxylation of (Pro-Pro Gly)l 0 in tile triple-helical conformation clearly demonstrated that the triple-helical conformation does not in itself prevent hydroxylation. The V for the helical form was 64% of the V for the random-coil form but the K m in molar concentrations was the same for both forms. Hydroxylation of the peptides Arg-Gly (Pro-Pro-Gly)~ and Glu-Gly-(ProPro Gly)~ indicated that the presence of a basic amino acid in the NH2-terminal end decreases the Km. Comparison of three more complex peptides indicated that modifications of amino acids in the - X - P r o Gly- triplets which are hydroxylated as well as modified in adjacent triplets can affect the reaction with protoeollagen proline hydroxylase.
INTRODUCTION The hydroxyproline in collagen is synthesized by the hydroxylation of proline which has been incorporated into peptide linkages. The peptidyl proline is hydroxylAbbreviations: Aoc-, acetoxycarbonyl; Boc-, tert-butyloxycarbonyl; OBzl-, benzyloxy; To-, tosyl. * Present address:Department of Medical Biochemistry, University ofOulu, Oulu, Finland. ** Address for reprint requests: Dr. D. J. Prockop, Department of Biochemistry, The Rutgers Medical School, New Brunswick, N.J.U.S.A. Biochim. Biophys. dcta, 271 (1972) 347-356
348
K.I. KIVIRIKKO dt a~.
ated either while nascent chains are being assembled on ribosomal complexes or after the complete assembly of a proline-rich and lysine-rich polypeptide precursor of collagen called protoeollagen (for review, see ref. I). The hydroxylating enzyme, protocollagen proline hydroxylase, has been purified from chick embryos 2 a and newborn rat skin 5, and it has been shown to require as co-factors or co-substrates (),~, Ire 2~ , aketoglutarate and a reducing substance such as ascorbate t. Protoeollagen proline hydroxylase does not hydroxylate tree proline but it hydroxylates proline in the " Y - p o s i t i o n ''6 s of repeating triplets ()f - X Y Gly in protocollagen and in synthetic peptides. No synthesis of hydroxyproline was observed when the tripeptide Gly Pro Pro was used as substrate for the enzyme :~ but a small rate of hydroxylation was obtained with the tripeptide Pro P r o - G l y ~°. Studies with a series of peptides with the general structure (X Pro (;ly)n demonstrated a large effect of chain length on the interaction of the enzyme in that longer peptides were hydroxylated much more readily than shorter peptides of the same structure~, ~° ~4. There are conflicting data, h o w e v e r , as to the optimal length of the peptide, it was initially reported that the i<~,~ for peptides of the structure (Pro Pro-Gly)n decreased as the average molecular weight was increased up to 4ooo (~or 8ooo ~2. Subsequently, however, one laboratory ~:~rel)orted that ( P r o - P r o Gly)l o with a molecular weight of about 26oo was hydroxylated at a higher rate than (Pro-Pro-Gly)20 and another laboratory ~° reported that the rate of hydroxylation with (Pro P r o 4 / l y ) s was greater than the rates observed with (Pro Pro (;ly)l o or (Pro Pro Gly)l s or (Pro Pro Gly)20. This discrepancy is probably explained by differences in conformation between the peptides employed in the experiments. The initial studies used peptides prepared with polymerization techniques which gave polydisperse products and which had a limited tendency to form collagen-like helical structures ~'~. The peptides used in the subsequent studies were synthesized with a new solid-state technique which gave homogeneous products x~ and which had a marked tendency to form triple-helical structures when the peptide contained Io or more tripletsXT, ~8. The formation of helical structures clearly complicates studies on the effect of peptide length, since (Pro-Pro-Gly)l,~ in a random-coil conformation was found to be hydroxylated more readily than the same peptide in a helical conformation ~°. Unfortunately the published datal°, ~a on the effect of chain length on the hydroxylation of (Pro Pro-Gly)n synthesized with a solid-state technique is based on experiments in which the ( P r o - P r o Gly)s was random-coil, the (Pro-Pro-(ilY)l 0 was partly triple-helical, and the (Pro--Pro-Gly)l 5 and (Pro Pro Gly)20 were either triple_tmlical mononlers or aggregates of triple_helical structures~7 ~.~ Some information is also available concerning the effect of variations in the amino acid sequences on the hydroxylation of proline in peptides. Poly-L-proline is not a substrate" for the enzyme and instead is a competitive inhibitor ~,12,'°. Peptides with the structure (Gly Pro (;ly)n are also not substrates but are competitive inhibitors for the enzyme 14. Studies with a series of analogues of the peptide horlnone bradykinin which has a sequence of Pro Pro G l y - showed that the peptide was a substrate ~ but the substrate activity was lost if the glyeine was replaced with some other amino acid "~2. These studies further showed that there were significant changes in Km and V if the proline in the "X-position" was replaced by other amino acids or if amino acid sequences adjacent to the - P r o - P r o Gly-triplet were varied. In the present work a series of peptides containing sequences of - X - P r o (ily were synthesized b y a solid-state method which gave homogeneous products. These Biochim. Biophys. Acfa, z 71 (~07~'1 347-350
PROTOCOLLAGEN PROLINE HYDROXYLASE
349
peptides were then used to study the synthesis of hydroxyproline by protocollagen proline hydroxylase in order to further examine the effect of polypeptide length, helical conformation and amino acid sequence on the reaction. MATERIALS AND METHODS
Synthetic peptides Ttle synthetic peptides used here were synthesized at the Peptide Center of the Institute for Protein Research, Osaka University, Osaka, Japan. The polytripeptides (Pro-Pro-Gly)5, (Pro-Pro-Gly)10, (Pro-Pro-Gly)l 5 and (Pro-Pro Gly)20 were synthesized by a specific modification of the Merrifield procedure for solid-state synthesis 16. As reported previously, the peptides were homogeneous by several criteria 16-19. The A r ~ G l y - ( P r o Pro Gly)5 was synthesized by coupling Aoc-Arg(To) Gly to (Pro-Pro-Gly)a-resin before cleaving the peptide off with H F in anisole. The HI; and neutral contaminants were removed by passing the peptide in water through an anion exchange resin (Dowex-I, X 2, OH- form). Tlle eluted sample was adjusted to pH 4 with acetic acid, lyophilized and then dried i~ vacuo over P205 at room temperature for 24 h. The peptide was homogeneous as measured by paper electrophoresis at pH 4.8 in 0.2 M acetate-pyridine buffer. The calculated amino acid analysis was Pro IO, Gly 6 and Arg I, and the observed values were Pro io.oo, Gly 5.63 and Arg 1.o2. The Glu-Gly-(Pro-Pro-Gly)a was synthesized by coupling first Aoc-Gly and then Boc-Glu(OBzl) to (Pro-Pro Gly)a-resin before cleaving the peptide from the resin. The H F was removed by passing the peptide in water through a column of Amberlite IR-45 (acetate form) and the eluted sample was lyophilized. The peptide was then dried as described above. The peptide was homogeneous when exanlined by paper electrophoresis at pH 4.8 in 0.2 M acetate-pyridine buffer. The calculated amino acid analysis was Pro IO, Gly 6, and Glu I, and the found values were Pro IO.OO, Gly 5.97 and Glu I.OO. The peptide Arg Gly-(Leu-Pro-Gly)5 was synthesized by step-wise coupling of Aoc-Leu Pro Gly with the same procedure used to synthesize (Pro-Pro-Gly)5. AocArg(To)-Gly was coupled to the (Leu-Pro-Gly)~-resin before cleaving the peptide off. The H F and neutral contaminants were removed by passing the peptide in water through a Dowex-I column (acetate form). The eluted sample was lyophilized and then chromatographed on a CM-Sephadex column (Pharmaeia C-25) with a gradient of I mM to 200 mM ammonium acetate, pH 5.5. Six peaks were obtained but 75% of the material was recovered in the major peak which was taken for further study. The calculated amino acid analysis for Arg-Gly-(Leu-Pro-Gly)~ was Pro 5, Gly 6, Leu 5 and Arg I and the observed values for the major peak were Pro 4.77, Gly 5.97, Leu 5.00 and Arg I.OO. The peptide Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Gly)4 was prepared by synthesizing (Pro-Pro-Gly)4-resin with the same procedures used for (Pro-Pro-Gly)n. Aoc-amino acids were coupled stepwise to the (Pro-Pro-Gly)4-resin and the peptide was then cleaved from the resin with HF. The peptide (Pro-ProGly)4-Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Gly)4 was prepared in the same manner except that before the peptide was cleaved from the resin, (Pro-ProGly)4 was added to the NH2-terminal end by repeated coupling with Aoc-Pro-ProGly. H F and neutral contaminants were removed by passing the peptides in water Biochim. Biophys..4cta, 271 (1972) 347-356
350
K . I . KIVIRIKKO ct al.
through an anion-exchange resin (Dowex I-XS) and the eluate was adjusted to pH 4 with acetic acid. The samples were lyophilized and dried at room temperature over P2Oa in i,aclto. The data on the purity of these two complex peptides were published previously and they indicated a small amount of heterogeneity2:L
E.n~vmatic reaction and assays Protocollagen proline hydroxylase was prepared as described by Halme ct al. 2 through the step of chromatography on DEAE-cellulose. The final preparation had a small amount of lysine hydroxylase activity but this activity was not sufficient to give more than a z% error in assaying the 14CQ released from a-~a4C]ketoglutarate ill the experiments on the hydroxylation of proline in the two complex peptides which contained both proline and lysine 2a. The enzymatic reaction under standard conditions "~awas carried out in a final volume of I.O ml which contained about 5 #g of the preparation of enzyme, o.o5 mM FeSO4, o. I mM a-[I-14C]ketoglutarate (obtained from Calbiochem and adjusted to a final specific activity of 35 ooo dpm per o.I #mole), 2 mM ascorbic acid, o. ~, nag of catalase (Calbiochem) ~, o.I mM dithiothreitol (Sigma) 24, 2 mg bovine serum albumin (Sigma) 24, and 50 mM Tris-HC1 buffer adjusted to pH 7.8 at 25 °C. The samples were incubated at 37 °(" for 2o rain and the ~4COa was trapped and counted with a minor modification 4 of the procedure of Rhoads and Udenfriend zS. All counting of 1~(; was t)erformed in an Intertechnique liquid scintillation spectrometer with an efficiency of 89% and a background of 25 cpm. Unless otherwise indicated, all peptide substrates were thermally denatured by heating to zoo °(" for Io min and quenching to 4 °C immediately before they were added to the incubation system. RESULTS
Effects of chain length on the hydro.Lvlation of random-coil forms of (Pro-Pro-G@),, In order to examine the effects of chain-length on the synthesis of hydroxyproline, the peptides (Pro-Pro-Gly)n with n = 5, io, 15 and 20 were thermally denatured and then incubated with protocollagen proline hydroxylase. The incubation conditions were adjusted so that initial velocity of the reaction was measured. Double reciprocal plots of initial velocity and substrate concentration were found to be linear over relatively broad range (Fig. I), even though with the three largest peptides deviations from linearity were observed with concentrations exceeding the Km (not shown). The plots indicated that the V was the same for all four peptides but the Km decreased as n was increased from 5 to 15 (Table I). No significant difference in Km was observed between (Pro--Pro-Gly)l.~ and (Pro-Pro Gly)20 but exact determinations of the Km were difficult with these peptides because the values were too low for accurate assay. It should be noted also that the Km values of zo-I 5 #g/ml obtained here with (Pro-Pro Gly)a a and (Pro Pro-Gly)20 are one order of magnitude less than the values of zoo /,g/ml 6 or z4o-25o/,g/mP 2 previously reported for the heterogeneous polymers of (Pro-Pro-Gly)n of comparable molecular weights.
Effect of triplc-hdical conformation on the hydro.Lvlation of (Pro-Pro-Gly)l o Kikuchi ct al. 1° reported that the random-coil form of (Pro-Pro-Gly)l,~ synthesized with the solid-state technique was hydroxylated at a more rapid rate than the l~iochim, l~b@hys. Achz, 27t (~97 z) 347 356
PROTOCOLLAGEN PROLINE HYDROXYLASE
~5 1
ON 4 (.1
7 2
0
I -;'0
20
40
60
80
I00
, 120
= 140
l 160
2. s
Fig. i. Double reciprocal plots of s u b s t r a t e c o n c e n t r a t i o n a n d t h e initial velocity for t h e h y d r o x y lation of ( P r o - P r o - G l y ) n . T h e e n z y m i c reaction was carried o u t as described in t h e t e x t a n d t h e e v o l u t i o n of [141C()2 from a-[I-]4C~lketoglutarate was m e a s u r e d . E a c h v a l u e s h o w n is t h e m e a n of d u p l i c a t e sanlples. T h e s u b s t r a t e c o n c e n t r a t i o n is e x p r e s s e d in m g / m l a n d t h e a m o u n t of ]4CO.2 is e x p r e s s e d in cpm. (_;, ('), h y d r o x y l a t i o n of (Pro Pro Gly)a; O - - O , h y d r o x y l a t i o n of (Pro - P r o - ( ; l y ) , o. TABLE I EFFECT
OF C H A I N L E N G T H
BY PROTOCOLLAGEN
ON T H E
PROLINE
HYDROXYLATION
OF R A N D O M - C O I L
FORMS OF ( P r o - ] ) r o - ( ~ l y ) n
HYDROXYLASE
T h e p e p t i d e s with t h e s t r u c t u r e ( P r o - P r o - G l y ) n were d e n a t u r e d a n d h y d r o x y l a t e d with protocollagen proline h y d r o x y l a s e as described in t h e text. T h e values s h o w n are t h e m e a n of at least t h r e e s e p a r a t e e x p e r i m e n t s (for typical e x a m p l e of double reciprocal plots, see Fig. 1).
Peptide substratc
(Pro-Pro-Gly)~ ( P r o - P r o - G l y ) j0 (Pro-i~ro-Gly)15 (Pro-Pro-Gly)2~, RCM*** Protocollagen***
l?elatiw~ I" (°o)
If,,, (/~g/ml)
(ftM Y-Pro*)
(/*M peptide)
lOO lOO l°5 lO 5
45 ° 7° [o-I5"* [o-I5"* IO I
18oo 28o 4°-6°'* 4o_6o ** 15 I
35 ° 3o 3 4"* 2_3** o.2 O.Ol
* Values indicate K,,, expressed in t e r m s o f / , M c o n c e n t r a t i o n of prolyl residues w h i c h are in t h e Y-position of r e p e a t i n g - X Y - G l y - triplets in t h e s u b s t r a t e . In t h e case of RCM, t h e prolyl r e s i d u e s in t h e Y-position are a s s u m e d to be o n e - h a l f of t h e 17o prolyl residues per p o l y p e p t i d e chainaL ** Values too low for a c c u r a t e assays. *** RCM is r e d u c e d a n d c a r b o x y m e t h y l a t e d collagen from t h e cuticle of Ascaris lumbricoides. T h e /fro v a l u e s for RCM 36 a n d protocollagen 38 are t a k e n from p r e v i o u s publications.
triple-helical form but they did not determine
the effects of helical conformation
on
K m a n d V. I n t h e e x p e r i m e n t s
reported here (Pro-Pro-Gly)l 0 was used to examine t h e i n f l u e n c e o f c o n f o r m a t i o n s i n c e i t is t h e s h o r t e s t p e p t i d e o f t h e s e r i e s n = 5, IO, I 5 a n d 2o w h i c h f o r m s t h e t r i p l e - h e l i c a l s t r u c t u r e i n a q u e o u s s o l u t i o n l 6 , 1 7 . T h e l o n g e r
peptides
n =
I5 and 2o were not studied
because triple-helical forms were less solu-
blelG 19 a n d b e c a u s e p r e v i o u s s t u d i e s d e m o n s t r a t e d that they aggregated into microcrystalline segments at temperatures s u i t a b l e f o r t h e e n z y m i c r e a c t i o n 10.
Biochim. Biophys. Acta, 271 (1972) 347-356
352
K . I . KIVIRIKKO (~t. a]. ,
i
,
i
,
t
,
6 oN o
g
10
t9
v
4
2
o
o
5 2
t -ZO
0
20
i 40
i 60 I
I 80
I I00
I 120
I 140
1 160
o
-4
t
,
t
4
8
12
,
,
i
i
,t
16
20
24
Z8
32
I
Fig. 2. Double reciprocal plots of s u b s t r a t e c o n c e n t r a t i o n a n d initial velocity for t h e h y d r o x y l a t i o n of (Pro Pro-Gly)~ 0 in t h e r a n d o m - c o i l f o r m a n d t h e triple-helical form. T h e h y d r o y x l a t i o n was carried o u t as described u n d e r M e t h o d s u n d e r s t a n d a r d conditions e x c e p t t h a t t h e reaction was clone at ~5 °C because t h e triple-helical f o r m begins to m e l t at h i g h e r t e m p e r a t u r e s . T h e v a l u e s are e x p r e s s e d as described in Fig. i. T h e Km for t h e helical form was 1 8 o / t g / m l ~md for t h e randora-coil form was 6o/,g/lnl. C3--C), h y d r o x y l a t i o n of (Pro-Pro-Gly)xo in t h e n a t i v e tripled~elical f o r m ; Q - - O , h y d r o x y l a t i o n of (Pro-Pro-Gly)10 in t h e r a n d o m - c o i l form. Fig. 3- D o u b l e reciprocal plots of s u b s t r a t e c o n c e n t r a t i o n a n d initial velocity for t h e h y d r o x y l a tion of Arg G l y - ( P r o Pro Oly).~, a n d G l u - G l y (Pro-Pro-Gly)5. T h e reaction conditions were as described in M e t h o d s a n d t h e values are e x p r e s s e d as s h o w n in Vig. i. O - - O , h y d r o x y l a t i o n of Arg._Gly_(Pro_Pro_Gly)a ; re__m, h y d r o x y l a t i o n of Glu Gly (Pro Pro-Gly).~; (~ ( , h y d r o x y l a t i o n of (Pro Pro-Oly).~.
In order to convert the (Pro-Pro-Gly)l 0 to the random-coil form the peptide was denatured by heating to ioo °C for IO min and was quenched at 4 °C for 3 rain immediately before adding to the incubation system. The hydroxylation of both the native and the denatured peptide was then carried out at 15 °C because the triplehelical structure begins to melt at higher temperatures ~G ~s. The Km for the helical form was I8o/tg/ml and it was 60 #g/ml for the random-coil form (Fig. 2). There was a significant effect on the V in that the value for the helical form was 64% of the value for the random-coil form.
Edy?ct of variations in NH~-terminal addition, s and variations in amino acid seque,nce o~z the ]kvdro.Lvlation of (X-Pro-Gly) In order to examine the effects of positively and negatively charged amino acids on the hydroxylation of (Pro-Pro-Gly)5, the peptides Arg-Gly-(Pro Pro-Gly)~ and G l u - G l y - ( P r o - P r o Gly)5 were synthesized. The peptide Arg-Gly (Pro-Pro Gly)~ was a better substrate than G l u - G l y - ( P r o - P r o Gly)~ in that the V was slightly higher and the Km was about one-half (Fig. 3 and Table II). In order to examine the effects of varying the amino acid in the X-position of (X-Pro-Gly)5, the peptide Arg-Gly-(Leu-Pro-Gly)5 was synthesized. Comparison of Arg-Gly-(Pro-Pro-Gly)5 with Arg-Gly-(Leu-Pro-Gly)5 indicated that the presence of leucine in the X-position had no effect on the Km but the V of the leueine-containing peptide was about 65°,/0 of the V for Arg-Gly-(Pro-Pro-Gly)5 (Table II). Biochim. Biophys. dcta, 27 r
(I972) 347-356
353
PROTOCOLLAGEN PROLINE HYDROXYLASE
TABLE II EFFECT
OF VARIATIONS
IN ~H,z-TERMINAL
ADDITIONS
AND
VARIATIONS
IN AMINO ACID SEQUENCE
(X-Pro-Gly)~ B v P R O T O C O L L A G E N P R O L I N E H Y D R O X Y L A S E The peptides were denatured and hydroxylated with protocollagen proline hydroxylase as described in the text. The values shown are the mean of two or three experiments {for typical example of double reciprocal plots, see Fig. 2). ON THE
HYDROXYLATION
OF
Peptide substrate
(Pro-i~ro-Gly)~ Arg-Gly--(Pro-Pro-Gly).~ Ghl-Gl_v-(Pro-Pro-G1 y)> Arg-Gly-(Leu-l'ro-Gly)~
Relative V (%)
1(~,~ @g/ml)
(tt..]l pcptide)
r oo
45o
35o
I 15
250
17 °
5s° 25°
4°o I6O
95 75
Comparison of ( P r o - P r o - G l y ) u with more complex peptides Since the h y d r o x y l a t i o n of proline in protocollagen presents a more complex situation t h a n t h a t presented b y a n y of the peptides e x a m i n e d above, two more con:plex peptides were studied (Table III). The V for the two complex peptides was the same a n d significantly greater t h a n the value for (Pro-Pro-Gly)5 a n d ( P r o - P r o - G l y ) : 0. The K m for the longer peptide was less t h a n the value for the shorter peptide, demons t r a t i n g the effect of chain length even in these complex peptides. It should be noted t h a t the peptide with one sequence of ( P r o - P r o - G l y ) , had a lower Km t h a n ( P r o - P r o Gly)5 a n d therefore it was a better substrate t h a n one might expect for (Pro-Pro-Gly)4 alone. Also, the peptide with two sequences of (Pro--Pro-Gly)4 had a lower Km t h a n one would expect for (Pro-Pro-Gly)s. TABLE lII HYDROXYLATION
OF TWO COMPLEX
PEPTIDES
BY" P R O T O C O L L A G E N
PROLINE
HVDROXYLASE
The peptides were denatured and hydroxylated with protocollagen proline hydroxylase as described in the text.
Peptide substrate
(Pro-Pro-Gly)~ (Pro-Pro-Gly)10 Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Oly) 4 (Pro-Pro-Gly)4-Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pr°-Pro-GlY)4
Number of amino acid residue in peptide
Number of -Pro-Pro-Gly/riHets i~z peptide
Relativ~ V (°.o)
15 30
5 IO
21 33
Km (t~g/ml)
(ltM peptide)
1oo IOO
45° 7°
35o 3o
4
160
2oo
I oo
8
10o
lOO
~o
DISCUSS/ON The h y d r o x y l a t i o n of p e p t i d e - b o u n d proline b y protocollagen proline h y d r o x y lase d u r i n g the biosynthesis of collagen presents an u n u s u a l l y complex situation. A b o u t a h u n d r e d prolyl residues in each polypeptide chain m u s t be h y d r o x y l a t e d a n d alt h o u g h a single productive e n c o u n t e r between the enzyme a n d the polypeptide sub-
Biochim. Biophys. Acta, 27:(1972 ) 347-356
354
K.I. KIVIRIKKOel al.
strate involves a considerable length of the substrate 26, multiple encounters are required to complete the hydroxylation 27. Also, it has been demonstrated that as the hydroxylation of the polypeptide chains proceeds, the affinity of the enzyme for the substrate decreases markedly 27. These observations in part help to explain the observation that in some 2s-a° but not all al collagens recovered from extracellular fibers the prolyl residues in specific "Y-positions" are incompletely hydroxylated. Hydroxylation of the peptides (Pro-Pro-Gly)n in tile series n - 5, Io, 15 and 20 indicated that if the peptides are examined in the random-coil form, there is no difference among the peptides in 17, but there is a marked effect of chainqength on tile Km value. The results differ trom those previously obtained with homogeneous peptides of tile same series~°, ~a and the difference is probably explained by the fact that in the earlier studies the peptides were not all in the same conformation (see Introduction). The results are consistent with the observations made previously with heterogeneous polymers of about the same structure6,*1, ~ but the Km values observed here are at least one order of nlagnitude less. However, even the lowest Km values obtained here with (Pro-Pro-Gly)l 5 and (Pro-Pro-Gly)20 are still higher than the values obtained previously with reduced and carboxymethylated collagen from the cuticle of Ascaris or with the natural substrate for the enzyme, protocollagen (Table I). The lower Km value obtained with the latter substrates may in part be explained by the fact that their polypeptide chains are much larger. However, the data obtained with synthetic peptides in which the amino acid sequences were varied (see below) suggest that the low Km for protocollagen is in part explained by the presence of more complicated amino acid sequences in the polypeptide chains. Hydroxylation of (Pro-Pro-Gly)l 0 in the triple-helical conformation clearly demonstrates that triple-helical conformation does not in itself prevent hydroxylation. The I" for the helical form was 6430 of the V for the random-coil form and the Km for the triple-helical form was about three times the value for the random-coil form if the values are expressed on the basis of #g/ml. However, if account is taken of the fact that the molar concentration of peptide is reduced to one-third when the random-coil is converted to the triple-helical form, the Km values for the two forms are the same. This observation suggests that the affinity of the enzyme ti)r substrate molecules does not change when the random-coil chains are converted to triple-helical structures. It will be important however to verify this conclusion with additional peptides that form triple-helical structures under conditions appropriate for the enzymatic reaction and that are more soluble than (Pro-Pro Gly)l 5 and (Pro-Pro-Gly)20. The hydroxylation of (Pro-Pro-Gly)l o in a triple-helical conformation is consistent with previous results suggesting that protocollagen can be hydroxylated in both the native and random-coil forms. Only #g quantities of impure protocollagen preparations have been available for study, but three laboratories have reported either the same rate of hydroxylation with native and heat denatured pr()tocollagen a2 or a slightly higher rate with the native fornlaa, a4` However, tile results obtained with both (Pro Pro~ (;ly)~ 0 and protocollagen are not readily reconciled with the recent report concerning the further hydroxylation of proline in collagen extracted from extracellular fibers aS. When collagen from rat-tail tendon and other tissues was incubated with large anlounts of protocollagen proline hydroxylase, the hydroxyproline content was increased by 5-I5°/g but this further hydroxylation was not observed unless the collagen was thermally denatured. This observation might be explained if it is assumed I~iochi*~,. I~iopll3,.~. A eta, -'71 (r97_,) 347-356
PROTOCOLLAGEN PROLINE HYDROXYLASE
355
that triple-helical conformation does not have much effect on the initial hydroxylation of protocollagen but it significantly retards the hydroxylation of the last few prolyl residues in the "Y-position" of the molecule. In particular, it is possible that specific prolyl residues in the Y-position are inaccessible to the enzyme because of steric hinderance from amino acids with long side chains in the other two a-chains of the triple-helix. Another observation which is not readily reconciled with the results obtained here is the inability to hydroxylate the collagen extracted from Ascaris cuticle unless it is denatured ~6. However, physical measurements on this collagen suggest that its conformation differs from that of other collagens 37. Hydroxylation of the peptide Arg-Gly-(Pro-Pro-Gly)5 and G l u - O l y - ( P r o Pro-Gly)5 indicated that the Km is lower and the V is slightly higher when the basic amino acid is present in the NHe-terminal end of the peptide. This result is consistent with observations that protocollagen proline hydroxylase is a highly acidic protein a. Further comparisons of peptides in this series indicated that substitution of leucine for proline in the X-position of the peptide A r g - G l y - ( X Pro-Gly)~, decreased the V by about one-third but had no effect on the P2m. The results as a whole emphasize that modifications of amino acids in the - X - P r o - G l y - triplets which are hydroxylated as well as modifications in adjacent triplets can affect the reaction with protocollagen proline hydroxylase. This conclusion is further supported by observations made with more complex peptides. The V values obtained with these peptides were the highest in the series studied here, and this effect is explained by the presence of the three complex triplets not containing proline but rich in basic amino acids. The effect of chain length on Km was also seen in these peptides, since on a molar basis the Km for the longer peptide was one-third of that for the shorter peptide.
ACKNOWLEDGMENTS
Tile authors gratefully acknowledge the expert technical assistance of Mrs Anita Cywinski. This work was supported in part by N.I.H. grants GRS-7IO6, F R - I o 7 and AM-14,526 from the U.S. Public Health Service. REFERENCES I M. E. G r a n t a n d D. J. Prockop, N. Engl. J. ~lTed., 286 (i972) 194,242, 291. 2 J. H a l m e , K. 1. K i v i r i k k o a n d K. Simons, Bioehim. Biophys. Acla, 198 (197 o) 460. 3 M. PS,nk~ilS.inen, H. Aro, K. S i m o n s a n d K. I. Kivirikko, Biochim. Biophys. Acta, 221 (197 o) 559. 4 R. A. Berg a n d D. J. Prockop, s u b m i t t e d for publication. 5 R. E. R h o a d s a n d S. Udenfriend, Arch. Biochem. Biophys., 139 (197 o) 329. 6 K. i. Kivirikko a n d D. J. Prockop, J, Biol. Chem., 242 (1967) 4007 . 7 J. J. H u t t o n , A. K a p l a n a n d S. Udenfriend, Arch. Biochem. Biophys., 121 (1967) 384 • 8 A. N o r d w i g a n d F. K. Pfab, Biochim. Biophys. Acla, 181 (1969) 52. 9 K. I. t£ivirikko a n d D. J. Prockop, Arch. Biochem. Biophys,, 118 (1967) 611. lO Y. Kikuchi, D. Fu.jimoto a n d N. T a m i y a , Biochem. J., 115 (1969) 569. t i D. J. Prockop, K. J u r a a n d J. Engel, Z. Physiol. Chem., 348 (1967) 553. 12 J. J. H u t t o n , A, Marglin, 13. W i t k o p , J, K u r t z , A. I3erger a n d S. Udenfriend, Arch. Biochem. Biophys., 125 (1968) 77913 F. S u z u k i a n d E. K o y a m a , Biochim. Biophys. Acta, 177 (1969) 154. 14 K. ]. Kivirikko, D. J. Prockop, G. P. Lorenzi a n d E. R. 131out, J. Biol. Chem., 244 (1969) 2755.
Biochim. Biophys. Acta, 27I (1972) 347-356
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I~. i. KIVIRIKKO Cl 6/1.
15 J. Engel, J, K u r t z , E. K a t c h a l s k i a n d A. Berger, J. 2~fol. Biog., t 7 (I966) 255. I6 S. S a k a k i b a r a , Y. Kishida, Y. Kikuchi, R. Sakai a n d K. K a k u c h i , Bull. Chem. Soc. Japa~, 41 {I968) I273. 17 Y. K o b a y a s h i , R. Sakai, K. K a k i u c h i a n d T. I s e m u r a , Biopo(vmers, 9 (I97o) 415 • i8 1~. A. Berg, B. 13. Olsen a n d D. J. Prockop, J. Biol. Chem., 245 (~97 o) 5759. 19 B. R. Olsen, R. A. Berg, S. S a k a k i b a r a , Y. K i s h i d a a n d D. J. Prockop, ./r. Mol. Bi(~l., 57 (~97 t) 589, 20 1~[. [. Kivirikko, V. (;anser, J. [~ngel a n d D. J. Prockop, Z. Physiog. Chem., 3,t~ (1947) ' 3 4 I . 2i R. [[. R h o a d s a n d S. Udenfriend, Arch. Biochem. Biophys., I33 (1969) 1o8. 22 J. O'D. McGee, R. E. R h o a d s a n d S. Udenfriend, Arch. Biochem. Biophys., I44 (~97~) 343. 23 K. I. Kivirikko, K. Shudo, S. S a k a k i b a r a &nd D. J. Prockop, Biochemist~T, ~t (I972) i22. 24 R. E. R h o a d s , J. J. H u t t o n a n d S. Udenfriend, Arch. Biochem. Biophys., 122 (1967) 8o5. 25 R. 1£. F, h o a d s a n d S. U d e n f r i e n d , Proe. Natl. Acad. Sci. U.S., 60 (I968) 1473. 26 1). J. P r o c k o p a n d K. I. Kivirikko, J . Biol. Chem., 244 (1969) 4S3,s. 27 K. J u v a a n d D. J. Prockop, J. Biol. Chem., 244 (1969) 6486. 28 P. B o r n s t e i n , Biochemistry, 0 (I967) 3o82. 29 W. T. B u t l e r a n d S. L. Ponds, Biochemistry, io (197 I) 2076. 3 ° G. Balain, E. M. Click a n d P. Bornstein, Biochemistry, Jo (~971) 447 o. 31 A. H. K a n g a n d J. (;ross, Biochemistry, 9 (197 o) 796. 32 A. N o r d w i g a n d F. K. Pfab, Biochim. Biophys. Acta, I54 (I968) 6o3. 33 J. J. H u t t o n , A. L. T a p p e l a n d S. Udenfriend, Arch. Biochem. Biophys., i t s (i967) 23L. 34 K. i. Kivirikko, H. J. B r i g h t a n d D. J. Prockop, Biochim. Biophys. Acta, i 5 i (1958) 55 s. 35 R. E. R h o a d s , S. (Menfriend a n d P. Bornstein, .[. Biol. Chem., 246 (197 I) 4138. 36 1). F u j i m o t o a n d D. J. Prockop, J. Biol. Chem., 243 (~9()8) 4~3 s. 37 O. W. McBride a n d W. F. H a r r i n g t o n , Biochcmist~3,, () (i967) 1484. 38 Is[, [. Kivirikko a n d D. J. Prockop, Proc. Nat/. Acad. Sci. U.S., 57 (I967) 782.
Biochim. Biophys. Acta, 27i (1972) 347-356
347
BBA 36173 H Y D R O X Y L A T I O N OF (X-PRO-GLY)n BY PROTOCOLLAGEN P R O L I N E HYDROXYLASE E F F E C T OF CHAIN LENGTH, H E L I C A L CONFORMATION AND AMINO ACID SEQUENCE IN T H E SUBSTRATE
KARI I. KIVIRIt(KO*, YASUO KISHIDA, SHUMPEI SAKAKIBARA AND I)AI{XV1N J. PROCKOP** Departments qf .,lledicine and Biochemistry, University of Pennsylvania and the Philadelphia General Hospital, Philadelphia, Pa. 191o4 (U.S.A.) and from the Peptide Center, Institute for Protein Research, Osaka University, Osaka (Japan)
(Received March 3ist, 1972)
SUMMARY
A series of peptides containing sequences o f - X - P r o - G l y - were synthesized by a solid-state method which gave homogeneous products. These peptides were then used to study the synthesis of hydroxyproline by protocollagen proline hydroxylase. Hydroxylation of random-coil forms of the series of peptides (Pro Pro-Gly)n with n = 5, IO, 15 and 20 indicated no difference among the peptides in V but the A'm decreased markedly with chain-length. Hydroxylation of (Pro-Pro Gly)l 0 in tile triple-helical conformation clearly demonstrated that the triple-helical conformation does not in itself prevent hydroxylation. The V for the helical form was 64% of the V for the random-coil form but the K m in molar concentrations was the same for both forms. Hydroxylation of the peptides Arg-Gly (Pro-Pro-Gly)~ and Glu-Gly-(ProPro Gly)~ indicated that the presence of a basic amino acid in the NH2-terminal end decreases the Km. Comparison of three more complex peptides indicated that modifications of amino acids in the - X - P r o Gly- triplets which are hydroxylated as well as modified in adjacent triplets can affect the reaction with protoeollagen proline hydroxylase.
INTRODUCTION The hydroxyproline in collagen is synthesized by the hydroxylation of proline which has been incorporated into peptide linkages. The peptidyl proline is hydroxylAbbreviations: Aoc-, acetoxycarbonyl; Boc-, tert-butyloxycarbonyl; OBzl-, benzyloxy; To-, tosyl. * Present address:Department of Medical Biochemistry, University ofOulu, Oulu, Finland. ** Address for reprint requests: Dr. D. J. Prockop, Department of Biochemistry, The Rutgers Medical School, New Brunswick, N.J.U.S.A. Biochim. Biophys. dcta, 271 (1972) 347-356
348
K.I. KIVIRIKKO dt a~.
ated either while nascent chains are being assembled on ribosomal complexes or after the complete assembly of a proline-rich and lysine-rich polypeptide precursor of collagen called protoeollagen (for review, see ref. I). The hydroxylating enzyme, protocollagen proline hydroxylase, has been purified from chick embryos 2 a and newborn rat skin 5, and it has been shown to require as co-factors or co-substrates (),~, Ire 2~ , aketoglutarate and a reducing substance such as ascorbate t. Protoeollagen proline hydroxylase does not hydroxylate tree proline but it hydroxylates proline in the " Y - p o s i t i o n ''6 s of repeating triplets ()f - X Y Gly in protocollagen and in synthetic peptides. No synthesis of hydroxyproline was observed when the tripeptide Gly Pro Pro was used as substrate for the enzyme :~ but a small rate of hydroxylation was obtained with the tripeptide Pro P r o - G l y ~°. Studies with a series of peptides with the general structure (X Pro (;ly)n demonstrated a large effect of chain length on the interaction of the enzyme in that longer peptides were hydroxylated much more readily than shorter peptides of the same structure~, ~° ~4. There are conflicting data, h o w e v e r , as to the optimal length of the peptide, it was initially reported that the i<~,~ for peptides of the structure (Pro Pro-Gly)n decreased as the average molecular weight was increased up to 4ooo (~or 8ooo ~2. Subsequently, however, one laboratory ~:~rel)orted that ( P r o - P r o Gly)l o with a molecular weight of about 26oo was hydroxylated at a higher rate than (Pro-Pro-Gly)20 and another laboratory ~° reported that the rate of hydroxylation with (Pro P r o 4 / l y ) s was greater than the rates observed with (Pro Pro (;ly)l o or (Pro Pro Gly)l s or (Pro Pro Gly)20. This discrepancy is probably explained by differences in conformation between the peptides employed in the experiments. The initial studies used peptides prepared with polymerization techniques which gave polydisperse products and which had a limited tendency to form collagen-like helical structures ~'~. The peptides used in the subsequent studies were synthesized with a new solid-state technique which gave homogeneous products x~ and which had a marked tendency to form triple-helical structures when the peptide contained Io or more tripletsXT, ~8. The formation of helical structures clearly complicates studies on the effect of peptide length, since (Pro-Pro-Gly)l,~ in a random-coil conformation was found to be hydroxylated more readily than the same peptide in a helical conformation ~°. Unfortunately the published datal°, ~a on the effect of chain length on the hydroxylation of (Pro Pro-Gly)n synthesized with a solid-state technique is based on experiments in which the ( P r o - P r o Gly)s was random-coil, the (Pro-Pro-(ilY)l 0 was partly triple-helical, and the (Pro--Pro-Gly)l 5 and (Pro Pro Gly)20 were either triple_tmlical mononlers or aggregates of triple_helical structures~7 ~.~ Some information is also available concerning the effect of variations in the amino acid sequences on the hydroxylation of proline in peptides. Poly-L-proline is not a substrate" for the enzyme and instead is a competitive inhibitor ~,12,'°. Peptides with the structure (Gly Pro (;ly)n are also not substrates but are competitive inhibitors for the enzyme 14. Studies with a series of analogues of the peptide horlnone bradykinin which has a sequence of Pro Pro G l y - showed that the peptide was a substrate ~ but the substrate activity was lost if the glyeine was replaced with some other amino acid "~2. These studies further showed that there were significant changes in Km and V if the proline in the "X-position" was replaced by other amino acids or if amino acid sequences adjacent to the - P r o - P r o Gly-triplet were varied. In the present work a series of peptides containing sequences of - X - P r o (ily were synthesized b y a solid-state method which gave homogeneous products. These Biochim. Biophys. Acfa, z 71 (~07~'1 347-350
PROTOCOLLAGEN PROLINE HYDROXYLASE
349
peptides were then used to study the synthesis of hydroxyproline by protocollagen proline hydroxylase in order to further examine the effect of polypeptide length, helical conformation and amino acid sequence on the reaction. MATERIALS AND METHODS
Synthetic peptides Ttle synthetic peptides used here were synthesized at the Peptide Center of the Institute for Protein Research, Osaka University, Osaka, Japan. The polytripeptides (Pro-Pro-Gly)5, (Pro-Pro-Gly)10, (Pro-Pro-Gly)l 5 and (Pro-Pro Gly)20 were synthesized by a specific modification of the Merrifield procedure for solid-state synthesis 16. As reported previously, the peptides were homogeneous by several criteria 16-19. The A r ~ G l y - ( P r o Pro Gly)5 was synthesized by coupling Aoc-Arg(To) Gly to (Pro-Pro-Gly)a-resin before cleaving the peptide off with H F in anisole. The HI; and neutral contaminants were removed by passing the peptide in water through an anion exchange resin (Dowex-I, X 2, OH- form). Tlle eluted sample was adjusted to pH 4 with acetic acid, lyophilized and then dried i~ vacuo over P205 at room temperature for 24 h. The peptide was homogeneous as measured by paper electrophoresis at pH 4.8 in 0.2 M acetate-pyridine buffer. The calculated amino acid analysis was Pro IO, Gly 6 and Arg I, and the observed values were Pro io.oo, Gly 5.63 and Arg 1.o2. The Glu-Gly-(Pro-Pro-Gly)a was synthesized by coupling first Aoc-Gly and then Boc-Glu(OBzl) to (Pro-Pro Gly)a-resin before cleaving the peptide from the resin. The H F was removed by passing the peptide in water through a column of Amberlite IR-45 (acetate form) and the eluted sample was lyophilized. The peptide was then dried as described above. The peptide was homogeneous when exanlined by paper electrophoresis at pH 4.8 in 0.2 M acetate-pyridine buffer. The calculated amino acid analysis was Pro IO, Gly 6, and Glu I, and the found values were Pro IO.OO, Gly 5.97 and Glu I.OO. The peptide Arg Gly-(Leu-Pro-Gly)5 was synthesized by step-wise coupling of Aoc-Leu Pro Gly with the same procedure used to synthesize (Pro-Pro-Gly)5. AocArg(To)-Gly was coupled to the (Leu-Pro-Gly)~-resin before cleaving the peptide off. The H F and neutral contaminants were removed by passing the peptide in water through a Dowex-I column (acetate form). The eluted sample was lyophilized and then chromatographed on a CM-Sephadex column (Pharmaeia C-25) with a gradient of I mM to 200 mM ammonium acetate, pH 5.5. Six peaks were obtained but 75% of the material was recovered in the major peak which was taken for further study. The calculated amino acid analysis for Arg-Gly-(Leu-Pro-Gly)~ was Pro 5, Gly 6, Leu 5 and Arg I and the observed values for the major peak were Pro 4.77, Gly 5.97, Leu 5.00 and Arg I.OO. The peptide Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Gly)4 was prepared by synthesizing (Pro-Pro-Gly)4-resin with the same procedures used for (Pro-Pro-Gly)n. Aoc-amino acids were coupled stepwise to the (Pro-Pro-Gly)4-resin and the peptide was then cleaved from the resin with HF. The peptide (Pro-ProGly)4-Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Gly)4 was prepared in the same manner except that before the peptide was cleaved from the resin, (Pro-ProGly)4 was added to the NH2-terminal end by repeated coupling with Aoc-Pro-ProGly. H F and neutral contaminants were removed by passing the peptides in water Biochim. Biophys..4cta, 271 (1972) 347-356
350
K . I . KIVIRIKKO ct al.
through an anion-exchange resin (Dowex I-XS) and the eluate was adjusted to pH 4 with acetic acid. The samples were lyophilized and dried at room temperature over P2Oa in i,aclto. The data on the purity of these two complex peptides were published previously and they indicated a small amount of heterogeneity2:L
E.n~vmatic reaction and assays Protocollagen proline hydroxylase was prepared as described by Halme ct al. 2 through the step of chromatography on DEAE-cellulose. The final preparation had a small amount of lysine hydroxylase activity but this activity was not sufficient to give more than a z% error in assaying the 14CQ released from a-~a4C]ketoglutarate ill the experiments on the hydroxylation of proline in the two complex peptides which contained both proline and lysine 2a. The enzymatic reaction under standard conditions "~awas carried out in a final volume of I.O ml which contained about 5 #g of the preparation of enzyme, o.o5 mM FeSO4, o. I mM a-[I-14C]ketoglutarate (obtained from Calbiochem and adjusted to a final specific activity of 35 ooo dpm per o.I #mole), 2 mM ascorbic acid, o. ~, nag of catalase (Calbiochem) ~, o.I mM dithiothreitol (Sigma) 24, 2 mg bovine serum albumin (Sigma) 24, and 50 mM Tris-HC1 buffer adjusted to pH 7.8 at 25 °C. The samples were incubated at 37 °(" for 2o rain and the ~4COa was trapped and counted with a minor modification 4 of the procedure of Rhoads and Udenfriend zS. All counting of 1~(; was t)erformed in an Intertechnique liquid scintillation spectrometer with an efficiency of 89% and a background of 25 cpm. Unless otherwise indicated, all peptide substrates were thermally denatured by heating to zoo °(" for Io min and quenching to 4 °C immediately before they were added to the incubation system. RESULTS
Effects of chain length on the hydro.Lvlation of random-coil forms of (Pro-Pro-G@),, In order to examine the effects of chain-length on the synthesis of hydroxyproline, the peptides (Pro-Pro-Gly)n with n = 5, io, 15 and 20 were thermally denatured and then incubated with protocollagen proline hydroxylase. The incubation conditions were adjusted so that initial velocity of the reaction was measured. Double reciprocal plots of initial velocity and substrate concentration were found to be linear over relatively broad range (Fig. I), even though with the three largest peptides deviations from linearity were observed with concentrations exceeding the Km (not shown). The plots indicated that the V was the same for all four peptides but the Km decreased as n was increased from 5 to 15 (Table I). No significant difference in Km was observed between (Pro--Pro-Gly)l.~ and (Pro-Pro Gly)20 but exact determinations of the Km were difficult with these peptides because the values were too low for accurate assay. It should be noted also that the Km values of zo-I 5 #g/ml obtained here with (Pro-Pro Gly)a a and (Pro Pro-Gly)20 are one order of magnitude less than the values of zoo /,g/ml 6 or z4o-25o/,g/mP 2 previously reported for the heterogeneous polymers of (Pro-Pro-Gly)n of comparable molecular weights.
Effect of triplc-hdical conformation on the hydro.Lvlation of (Pro-Pro-Gly)l o Kikuchi ct al. 1° reported that the random-coil form of (Pro-Pro-Gly)l,~ synthesized with the solid-state technique was hydroxylated at a more rapid rate than the l~iochim, l~b@hys. Achz, 27t (~97 z) 347 356
PROTOCOLLAGEN PROLINE HYDROXYLASE
~5 1
ON 4 (.1
7 2
0
I -;'0
20
40
60
80
I00
, 120
= 140
l 160
2. s
Fig. i. Double reciprocal plots of s u b s t r a t e c o n c e n t r a t i o n a n d t h e initial velocity for t h e h y d r o x y lation of ( P r o - P r o - G l y ) n . T h e e n z y m i c reaction was carried o u t as described in t h e t e x t a n d t h e e v o l u t i o n of [141C()2 from a-[I-]4C~lketoglutarate was m e a s u r e d . E a c h v a l u e s h o w n is t h e m e a n of d u p l i c a t e sanlples. T h e s u b s t r a t e c o n c e n t r a t i o n is e x p r e s s e d in m g / m l a n d t h e a m o u n t of ]4CO.2 is e x p r e s s e d in cpm. (_;, ('), h y d r o x y l a t i o n of (Pro Pro Gly)a; O - - O , h y d r o x y l a t i o n of (Pro - P r o - ( ; l y ) , o. TABLE I EFFECT
OF C H A I N L E N G T H
BY PROTOCOLLAGEN
ON T H E
PROLINE
HYDROXYLATION
OF R A N D O M - C O I L
FORMS OF ( P r o - ] ) r o - ( ~ l y ) n
HYDROXYLASE
T h e p e p t i d e s with t h e s t r u c t u r e ( P r o - P r o - G l y ) n were d e n a t u r e d a n d h y d r o x y l a t e d with protocollagen proline h y d r o x y l a s e as described in t h e text. T h e values s h o w n are t h e m e a n of at least t h r e e s e p a r a t e e x p e r i m e n t s (for typical e x a m p l e of double reciprocal plots, see Fig. 1).
Peptide substratc
(Pro-Pro-Gly)~ ( P r o - P r o - G l y ) j0 (Pro-i~ro-Gly)15 (Pro-Pro-Gly)2~, RCM*** Protocollagen***
l?elatiw~ I" (°o)
If,,, (/~g/ml)
(ftM Y-Pro*)
(/*M peptide)
lOO lOO l°5 lO 5
45 ° 7° [o-I5"* [o-I5"* IO I
18oo 28o 4°-6°'* 4o_6o ** 15 I
35 ° 3o 3 4"* 2_3** o.2 O.Ol
* Values indicate K,,, expressed in t e r m s o f / , M c o n c e n t r a t i o n of prolyl residues w h i c h are in t h e Y-position of r e p e a t i n g - X Y - G l y - triplets in t h e s u b s t r a t e . In t h e case of RCM, t h e prolyl r e s i d u e s in t h e Y-position are a s s u m e d to be o n e - h a l f of t h e 17o prolyl residues per p o l y p e p t i d e chainaL ** Values too low for a c c u r a t e assays. *** RCM is r e d u c e d a n d c a r b o x y m e t h y l a t e d collagen from t h e cuticle of Ascaris lumbricoides. T h e /fro v a l u e s for RCM 36 a n d protocollagen 38 are t a k e n from p r e v i o u s publications.
triple-helical form but they did not determine
the effects of helical conformation
on
K m a n d V. I n t h e e x p e r i m e n t s
reported here (Pro-Pro-Gly)l 0 was used to examine t h e i n f l u e n c e o f c o n f o r m a t i o n s i n c e i t is t h e s h o r t e s t p e p t i d e o f t h e s e r i e s n = 5, IO, I 5 a n d 2o w h i c h f o r m s t h e t r i p l e - h e l i c a l s t r u c t u r e i n a q u e o u s s o l u t i o n l 6 , 1 7 . T h e l o n g e r
peptides
n =
I5 and 2o were not studied
because triple-helical forms were less solu-
blelG 19 a n d b e c a u s e p r e v i o u s s t u d i e s d e m o n s t r a t e d that they aggregated into microcrystalline segments at temperatures s u i t a b l e f o r t h e e n z y m i c r e a c t i o n 10.
Biochim. Biophys. Acta, 271 (1972) 347-356
352
K . I . KIVIRIKKO (~t. a]. ,
i
,
i
,
t
,
6 oN o
g
10
t9
v
4
2
o
o
5 2
t -ZO
0
20
i 40
i 60 I
I 80
I I00
I 120
I 140
1 160
o
-4
t
,
t
4
8
12
,
,
i
i
,t
16
20
24
Z8
32
I
Fig. 2. Double reciprocal plots of s u b s t r a t e c o n c e n t r a t i o n a n d initial velocity for t h e h y d r o x y l a t i o n of (Pro Pro-Gly)~ 0 in t h e r a n d o m - c o i l f o r m a n d t h e triple-helical form. T h e h y d r o y x l a t i o n was carried o u t as described u n d e r M e t h o d s u n d e r s t a n d a r d conditions e x c e p t t h a t t h e reaction was clone at ~5 °C because t h e triple-helical f o r m begins to m e l t at h i g h e r t e m p e r a t u r e s . T h e v a l u e s are e x p r e s s e d as described in Fig. i. T h e Km for t h e helical form was 1 8 o / t g / m l ~md for t h e randora-coil form was 6o/,g/lnl. C3--C), h y d r o x y l a t i o n of (Pro-Pro-Gly)xo in t h e n a t i v e tripled~elical f o r m ; Q - - O , h y d r o x y l a t i o n of (Pro-Pro-Gly)10 in t h e r a n d o m - c o i l form. Fig. 3- D o u b l e reciprocal plots of s u b s t r a t e c o n c e n t r a t i o n a n d initial velocity for t h e h y d r o x y l a tion of Arg G l y - ( P r o Pro Oly).~, a n d G l u - G l y (Pro-Pro-Gly)5. T h e reaction conditions were as described in M e t h o d s a n d t h e values are e x p r e s s e d as s h o w n in Vig. i. O - - O , h y d r o x y l a t i o n of Arg._Gly_(Pro_Pro_Gly)a ; re__m, h y d r o x y l a t i o n of Glu Gly (Pro Pro-Gly).~; (~ ( , h y d r o x y l a t i o n of (Pro Pro-Oly).~.
In order to convert the (Pro-Pro-Gly)l 0 to the random-coil form the peptide was denatured by heating to ioo °C for IO min and was quenched at 4 °C for 3 rain immediately before adding to the incubation system. The hydroxylation of both the native and the denatured peptide was then carried out at 15 °C because the triplehelical structure begins to melt at higher temperatures ~G ~s. The Km for the helical form was I8o/tg/ml and it was 60 #g/ml for the random-coil form (Fig. 2). There was a significant effect on the V in that the value for the helical form was 64% of the value for the random-coil form.
Edy?ct of variations in NH~-terminal addition, s and variations in amino acid seque,nce o~z the ]kvdro.Lvlation of (X-Pro-Gly) In order to examine the effects of positively and negatively charged amino acids on the hydroxylation of (Pro-Pro-Gly)5, the peptides Arg-Gly-(Pro Pro-Gly)~ and G l u - G l y - ( P r o - P r o Gly)5 were synthesized. The peptide Arg-Gly (Pro-Pro Gly)~ was a better substrate than G l u - G l y - ( P r o - P r o Gly)~ in that the V was slightly higher and the Km was about one-half (Fig. 3 and Table II). In order to examine the effects of varying the amino acid in the X-position of (X-Pro-Gly)5, the peptide Arg-Gly-(Leu-Pro-Gly)5 was synthesized. Comparison of Arg-Gly-(Pro-Pro-Gly)5 with Arg-Gly-(Leu-Pro-Gly)5 indicated that the presence of leucine in the X-position had no effect on the Km but the V of the leueine-containing peptide was about 65°,/0 of the V for Arg-Gly-(Pro-Pro-Gly)5 (Table II). Biochim. Biophys. dcta, 27 r
(I972) 347-356
353
PROTOCOLLAGEN PROLINE HYDROXYLASE
TABLE II EFFECT
OF VARIATIONS
IN ~H,z-TERMINAL
ADDITIONS
AND
VARIATIONS
IN AMINO ACID SEQUENCE
(X-Pro-Gly)~ B v P R O T O C O L L A G E N P R O L I N E H Y D R O X Y L A S E The peptides were denatured and hydroxylated with protocollagen proline hydroxylase as described in the text. The values shown are the mean of two or three experiments {for typical example of double reciprocal plots, see Fig. 2). ON THE
HYDROXYLATION
OF
Peptide substrate
(Pro-i~ro-Gly)~ Arg-Gly--(Pro-Pro-Gly).~ Ghl-Gl_v-(Pro-Pro-G1 y)> Arg-Gly-(Leu-l'ro-Gly)~
Relative V (%)
1(~,~ @g/ml)
(tt..]l pcptide)
r oo
45o
35o
I 15
250
17 °
5s° 25°
4°o I6O
95 75
Comparison of ( P r o - P r o - G l y ) u with more complex peptides Since the h y d r o x y l a t i o n of proline in protocollagen presents a more complex situation t h a n t h a t presented b y a n y of the peptides e x a m i n e d above, two more con:plex peptides were studied (Table III). The V for the two complex peptides was the same a n d significantly greater t h a n the value for (Pro-Pro-Gly)5 a n d ( P r o - P r o - G l y ) : 0. The K m for the longer peptide was less t h a n the value for the shorter peptide, demons t r a t i n g the effect of chain length even in these complex peptides. It should be noted t h a t the peptide with one sequence of ( P r o - P r o - G l y ) , had a lower Km t h a n ( P r o - P r o Gly)5 a n d therefore it was a better substrate t h a n one might expect for (Pro-Pro-Gly)4 alone. Also, the peptide with two sequences of (Pro--Pro-Gly)4 had a lower Km t h a n one would expect for (Pro-Pro-Gly)s. TABLE lII HYDROXYLATION
OF TWO COMPLEX
PEPTIDES
BY" P R O T O C O L L A G E N
PROLINE
HVDROXYLASE
The peptides were denatured and hydroxylated with protocollagen proline hydroxylase as described in the text.
Peptide substrate
(Pro-Pro-Gly)~ (Pro-Pro-Gly)10 Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pro-Pro-Oly) 4 (Pro-Pro-Gly)4-Ala-Arg-Gly-Met-Lys-Gly-His-Arg-Gly-(Pr°-Pro-GlY)4
Number of amino acid residue in peptide
Number of -Pro-Pro-Gly/riHets i~z peptide
Relativ~ V (°.o)
15 30
5 IO
21 33
Km (t~g/ml)
(ltM peptide)
1oo IOO
45° 7°
35o 3o
4
160
2oo
I oo
8
10o
lOO
~o
DISCUSS/ON The h y d r o x y l a t i o n of p e p t i d e - b o u n d proline b y protocollagen proline h y d r o x y lase d u r i n g the biosynthesis of collagen presents an u n u s u a l l y complex situation. A b o u t a h u n d r e d prolyl residues in each polypeptide chain m u s t be h y d r o x y l a t e d a n d alt h o u g h a single productive e n c o u n t e r between the enzyme a n d the polypeptide sub-
Biochim. Biophys. Acta, 27:(1972 ) 347-356
354
K.I. KIVIRIKKOel al.
strate involves a considerable length of the substrate 26, multiple encounters are required to complete the hydroxylation 27. Also, it has been demonstrated that as the hydroxylation of the polypeptide chains proceeds, the affinity of the enzyme for the substrate decreases markedly 27. These observations in part help to explain the observation that in some 2s-a° but not all al collagens recovered from extracellular fibers the prolyl residues in specific "Y-positions" are incompletely hydroxylated. Hydroxylation of the peptides (Pro-Pro-Gly)n in tile series n - 5, Io, 15 and 20 indicated that if the peptides are examined in the random-coil form, there is no difference among the peptides in 17, but there is a marked effect of chainqength on tile Km value. The results differ trom those previously obtained with homogeneous peptides of tile same series~°, ~a and the difference is probably explained by the fact that in the earlier studies the peptides were not all in the same conformation (see Introduction). The results are consistent with the observations made previously with heterogeneous polymers of about the same structure6,*1, ~ but the Km values observed here are at least one order of nlagnitude less. However, even the lowest Km values obtained here with (Pro-Pro-Gly)l 5 and (Pro-Pro-Gly)20 are still higher than the values obtained previously with reduced and carboxymethylated collagen from the cuticle of Ascaris or with the natural substrate for the enzyme, protocollagen (Table I). The lower Km value obtained with the latter substrates may in part be explained by the fact that their polypeptide chains are much larger. However, the data obtained with synthetic peptides in which the amino acid sequences were varied (see below) suggest that the low Km for protocollagen is in part explained by the presence of more complicated amino acid sequences in the polypeptide chains. Hydroxylation of (Pro-Pro-Gly)l 0 in the triple-helical conformation clearly demonstrates that triple-helical conformation does not in itself prevent hydroxylation. The I" for the helical form was 6430 of the V for the random-coil form and the Km for the triple-helical form was about three times the value for the random-coil form if the values are expressed on the basis of #g/ml. However, if account is taken of the fact that the molar concentration of peptide is reduced to one-third when the random-coil is converted to the triple-helical form, the Km values for the two forms are the same. This observation suggests that the affinity of the enzyme ti)r substrate molecules does not change when the random-coil chains are converted to triple-helical structures. It will be important however to verify this conclusion with additional peptides that form triple-helical structures under conditions appropriate for the enzymatic reaction and that are more soluble than (Pro-Pro Gly)l 5 and (Pro-Pro-Gly)20. The hydroxylation of (Pro-Pro-Gly)l o in a triple-helical conformation is consistent with previous results suggesting that protocollagen can be hydroxylated in both the native and random-coil forms. Only #g quantities of impure protocollagen preparations have been available for study, but three laboratories have reported either the same rate of hydroxylation with native and heat denatured pr()tocollagen a2 or a slightly higher rate with the native fornlaa, a4` However, tile results obtained with both (Pro Pro~ (;ly)~ 0 and protocollagen are not readily reconciled with the recent report concerning the further hydroxylation of proline in collagen extracted from extracellular fibers aS. When collagen from rat-tail tendon and other tissues was incubated with large anlounts of protocollagen proline hydroxylase, the hydroxyproline content was increased by 5-I5°/g but this further hydroxylation was not observed unless the collagen was thermally denatured. This observation might be explained if it is assumed I~iochi*~,. I~iopll3,.~. A eta, -'71 (r97_,) 347-356
PROTOCOLLAGEN PROLINE HYDROXYLASE
355
that triple-helical conformation does not have much effect on the initial hydroxylation of protocollagen but it significantly retards the hydroxylation of the last few prolyl residues in the "Y-position" of the molecule. In particular, it is possible that specific prolyl residues in the Y-position are inaccessible to the enzyme because of steric hinderance from amino acids with long side chains in the other two a-chains of the triple-helix. Another observation which is not readily reconciled with the results obtained here is the inability to hydroxylate the collagen extracted from Ascaris cuticle unless it is denatured ~6. However, physical measurements on this collagen suggest that its conformation differs from that of other collagens 37. Hydroxylation of the peptide Arg-Gly-(Pro-Pro-Gly)5 and G l u - O l y - ( P r o Pro-Gly)5 indicated that the Km is lower and the V is slightly higher when the basic amino acid is present in the NHe-terminal end of the peptide. This result is consistent with observations that protocollagen proline hydroxylase is a highly acidic protein a. Further comparisons of peptides in this series indicated that substitution of leucine for proline in the X-position of the peptide A r g - G l y - ( X Pro-Gly)~, decreased the V by about one-third but had no effect on the P2m. The results as a whole emphasize that modifications of amino acids in the - X - P r o - G l y - triplets which are hydroxylated as well as modifications in adjacent triplets can affect the reaction with protocollagen proline hydroxylase. This conclusion is further supported by observations made with more complex peptides. The V values obtained with these peptides were the highest in the series studied here, and this effect is explained by the presence of the three complex triplets not containing proline but rich in basic amino acids. The effect of chain length on Km was also seen in these peptides, since on a molar basis the Km for the longer peptide was one-third of that for the shorter peptide.
ACKNOWLEDGMENTS
Tile authors gratefully acknowledge the expert technical assistance of Mrs Anita Cywinski. This work was supported in part by N.I.H. grants GRS-7IO6, F R - I o 7 and AM-14,526 from the U.S. Public Health Service. REFERENCES I M. E. G r a n t a n d D. J. Prockop, N. Engl. J. ~lTed., 286 (i972) 194,242, 291. 2 J. H a l m e , K. 1. K i v i r i k k o a n d K. Simons, Bioehim. Biophys. Acla, 198 (197 o) 460. 3 M. PS,nk~ilS.inen, H. Aro, K. S i m o n s a n d K. I. Kivirikko, Biochim. Biophys. Acta, 221 (197 o) 559. 4 R. A. Berg a n d D. J. Prockop, s u b m i t t e d for publication. 5 R. E. R h o a d s a n d S. Udenfriend, Arch. Biochem. Biophys., 139 (197 o) 329. 6 K. i. Kivirikko a n d D. J. Prockop, J, Biol. Chem., 242 (1967) 4007 . 7 J. J. H u t t o n , A. K a p l a n a n d S. Udenfriend, Arch. Biochem. Biophys., 121 (1967) 384 • 8 A. N o r d w i g a n d F. K. Pfab, Biochim. Biophys. Acla, 181 (1969) 52. 9 K. I. t£ivirikko a n d D. J. Prockop, Arch. Biochem. Biophys,, 118 (1967) 611. lO Y. Kikuchi, D. Fu.jimoto a n d N. T a m i y a , Biochem. J., 115 (1969) 569. t i D. J. Prockop, K. J u r a a n d J. Engel, Z. Physiol. Chem., 348 (1967) 553. 12 J. J. H u t t o n , A, Marglin, 13. W i t k o p , J, K u r t z , A. I3erger a n d S. Udenfriend, Arch. Biochem. Biophys., 125 (1968) 77913 F. S u z u k i a n d E. K o y a m a , Biochim. Biophys. Acta, 177 (1969) 154. 14 K. ]. Kivirikko, D. J. Prockop, G. P. Lorenzi a n d E. R. 131out, J. Biol. Chem., 244 (1969) 2755.
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