Proline hydroxylation by oxygen

Proline hydroxylation by oxygen

SttOI~T COMN[UNICATIONS 425 (vide infra). On the basis of these findings perturbation of the cinnamate ion spectrum b y the protein can be exctuded ...

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(vide infra). On the basis of these findings perturbation of the cinnamate ion spectrum b y the protein can be exctuded as a possible explanation of the high 2max observed in the kinetic experiments (Table iI). These experiments stress the need for the careful choice of buffers in nil studies concerned with the elucidation of the enzyme reaction mechanisms with active acyl compounds as substrates. Further evidence for this con° clusion is derived from the observations that Tris functions as a more effective acyl acceptor than water in the ~-chymotrypsin-catalyzed hydrolysis of p-nitrophenylacetate a and of 4-cfs-benzylidene-2-phenyl-2-oxazolin-5-one. 4 The inadequacies of the Tris buffers arising as a result of the reactivity of Tris above p H 7.o and its poor buffering capacity below p H 7.5 have recently been reported 5& This investigation was supported by research grants from National Research Council, Defense Research Board and Department of University Affairs, Ontario. Department of Chemistry, University of Waterloo, Waterloo (Canada)


M. L. B]~NDF,R AND ]L. T. KAISER, ] . Am. Chem. Soc., 84 (I962) 2556. M. L. B]~NDER, G. IR. SC~IONBAUN AND B. ZERNER, ] . A m . Chem. Soc., 84 (-~962) 2540. L. ]FALLXR AND J. M. STUP,TEVAI,~% ]. Biol. Chem., 24I (I966) 4825. J. D~J:~Rs~,Y AND B. Z:m~NEP,, Biochem. Biophys. Res. Commun., 28 (i967) i73. N. E. GooD, G. D. WlNGET, W . %tINTER, T. N. CONNOLY, S. IZAWA AND t~. ~'~. ~[. SINGH, Biochemistry, 5 (~966) 467 . 6 M. PAVLI~, Biochim. Biophys. Acts, I39 (~967) I33.

I 2 3 4 5

Received December 28th, I967 * O n leave of a b s e n c e f r o m t h e U n i v e r s i t y of Salford, E n g i a n d .

Bioehim. Biophys. Acts, z56 (I968) 422-425

BBA 23 393

Proline hydroxylation by oxygen A non-enzymic system for lhe hydroxylation of aromatic molecules I-a, first described b y UDENF~IEN> et al.4,~ and later shown by CHVAPIL AND HURYCt{~ to oxidize proline to hydroxyproline, containing Fe e+, ascorbic acid and ethyienediaminetetraacetic acid (EDTA), and using either H20 2 or 02 as oxidant, has often been considered as a model for the hydroxylation of proiine to hydroxyproline in collagen formation. This is based on the fact that specific collagen proline residues in vivo are hydroxyIated enzymatically in a process where 0 2 becomes incorporated v,s, ascorbic acid and Fe e+ play important roles 9-12 and the involvement of free radicals has been suggestedla,74. In the present study an attempt has been made to better our understanding of the mechanism ira vivo by study of the conditions for proline hydroxylation in vitro, particularly b y comparing and contrasting the reactions with H~Oa and O~ in the Udenfriend-Chvapil system. The results presented in the preliminary report indicate that whereas hydroxylation b y H202 appears to be consistent with a free-radical Biochim. Biophys. Acts, i56 (I968) 425-428



mechanism, hydroxylation b y 02 involves neither the formation H202 nor free radicals. The basal reaction mixture routinely contained in a final volmne of 3.6 ml, the following reactants (in /,moles): F e S Q , 44; EDTA, I5; citric acid, 3oo; and Na2HPO4, I5o. The p H was adjusted to p H 4 or 6 as required. For the H202 reaction the basal medium was supplemented (in /mloles) with proline, 9; and H202, 880; in an atmosphere of N 2 or air. In the O 2 reaction, proline, 44; ascorbic acid, I62; and 0 2 (bubbled in as O 2 or air) for 15 rain. Other reactants were added as required. The reactions were routinely carried out at 4 o0 for 15 min. Hydroxyproline was determined b y the method of WOESSNER15. Hydroxy/ation by H202. A series of experiments have been carried out with H202 as the hydroxylating agent aimed at showing that, in fact, the hydroxylation of proline in this system is a free radical reaction. (I) Reaction mixtures as described were supplemented with hydroquinone (IO ~moles) or with reduced glutathione (equimolar with ascorbic acid), both of which are inhibitors of free radical reactions. The former inhibited hydroxylation by more than 99 % and the latter by more than 9 ° %. (2) The addition of Fe 2+ beyond the optimum would be expected to decrease the amount of hydroxyproline formed presumably b y favoring electron transfer from Fe 2+ to OH" before the latter can react with proline. This is in fact what is observed, and with a quadrupling of the Fe 2+ concentration hydroxylation is lowered b y about 50 %. (3) The only absolute requirements for the reaction have been found to be Fe 2+ and H202; these together constitute Fenton's reagent which is generally considered to hydroxylate organic molecules by a free radical reaction involving OH" as the active species 16. There was no significant difference in the degree of hydroxylation when the reaction was carried out either in air or under N 2. However, if O 2 was bubbled through the system, with certain reductants present, hydroxylation was significantly increased, suggesting that independent hydroxylation might be occurring in addition to that effected b y H202. Hydroxylation by 02. The 02 reaction has been studied under a variety of conditions ('Fable I). The reaction proceeds equally well when the 02 is supplied as air or the pure gas. I t has been found to have an absolute requirement for a reducing agent such as ascorbie acid at all Fe 2+ concentrations studied. Ascorbic acid can be replaced b y other reducing agents such as Fe(CN)63- or Fe(CN)a 4-. The yield of hydroxyproline is increased b y the addition of a less than stoiehiometrie amount of Fe(CN)68- or Fe(CN)~ 4-, to the complete system. Between p H 5 and 6, these con]pounds nearly triple the yield when present in the reaction mixture together with Fe 2+ and aseorbic acid. Above p H 5, hydroxylation of nearly the same intensity as t h a t obtained with Fe 2+ and ascorbic acid, is found with a reaction mixture containing Fe 2+ and Fe(CN), a- or Fe(CN), 4-, but no ascorbic acid. When H202 is the oxidant, 2.8 M Fe(CN)6 a- does not affect the yield. In contradistinction to the hydroxylation by H~O 2 proline hydroxylation by O~ is not inhibited b y hydroquinone or reduced glutathione, nor is it affected b y a 5-fold increase in Fe e+ concentration in the reaction mixture. An unusual characteristic of the reaction with 02 which sets it apart from that with H20~ is that under optimum conditions for the 02 reaction, only 0.6 to 0.7 % of proline initially present is converted to hydroxyproline. The hydroxyproline yield is kept low through a curious inhibition to further hydroxylation by O 2 which sets Biochim. Biophys. Acts, I56 (1968) 425-428



TABLE I YIELD OF HYDROXYPROLINE IN THE 0~ REACTION " C o m p l e t e " reaction m i x t u r e as described in t e x t at p H 4 and w i t h o u t I
Variation in conditions


Complete Fe ~+ omitted Ascorbic acid o m i t t e d Io #moles KaFe(CN)6 added, p H 6 i o #moles K~Fe(CN h added, p H 4 IO ~moles KaFe(CN)s added, p H 6, ascorbic acid o m i t t e d Io #moles h y d r o q u i n o n e added Lowered ascorbic acid (54 #moles) Lowered ascorbic acid (54 #moles), 54 ~moles GSH added I56 #moles Fe e+ added 44/~mo]es Fe > and 54 #moles ascorbic acid added after i o miD O~ F d + o m i t t e d ; Fe > added after 5 rain O~ Proline o m i t t e d ; proline added after 5 mid O 2

~ i. 8 * o I 30.0** 28 7 ~o 6 5 !4 I5 ,~i o

* F o r this value, a s t a n d a r d deviation of n= 2.4 was c o m p u t e d based on ~7 replicates, ** A s t a n d a r d deviation of ± t.6 was c o m p u t e d based on 8 replicates.

in within about 5 mid of initiation of the reaction b y O> After completion of the reaction, the yield is only slight!y increased by replenishing those components that might be expected to have become ineffective through oxidation, such as Fe > and ascorbic acid. This inhibition is not due simply to an alteration in the F e > - F e a+ relationship or to oxidation of ascorbic acid since it can neither be relieved b y addition of K F which forms a highly insoluble complex with Fe a+, nor mimicked b y addition of dehydroascorbic acid and/or Fe a+. This characteristic of the 0 2 reaction has been directed toward determining whether 0 2 hydroxytation proceeds b y a mechanism similar to H20 ~ hydroxylation, and whether it involves H20 ~production on the basis t h a t were the reactions similar, or ioo ~





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_~ 5oh zo~




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H 2 O2



Fig. I. H y d r o x y p r o l i n e formed b y K20~ in a s y s t e m fully inhibited t o w a r d s h y d r o x y l a t i o n b y O v E x p e r i m e n t a l conditions are those described in t e x t A , O~-inhibited reaction m i x t u r e s ; A, noninhibited controls. (The p l o t t e d h y d r o x y p r o t i n e values for the inhibited reaction h a v e been corrected b y s u b t r a c t i n g the h y d r o x y p r o l i n e f o r m e d initially b y O~.)

Biochim. Biophys. Acta, 156 (I968) 425-428



were H202 formation involved, the O2-inhibited reaction should not permit subsequent hydroxylation by added H202. To test this, a number oI tubes were charged with the basal reaction mixture containing both proline and ascorbic acid. O 2 was bubbled into half of these for 15 min. As the last entry in Table I shows, this is sufficiently long for complete inhibition of further hydroxylation by 02. An aliquot of 3o % H202 was then added to each tube and O 2 treatment carried out for another r 5 rain. Total yields of hydroxyproline, corrected for hydroxylation by O 2 in tubes with initial O 2 treatment, are shown in Fig. I. Clearly, prior O~ treatment does not markedly inhibit subsequent hydroxylation by H202. The reaction conditions in the two sets of tubes are not strictly comparable and the somewhat lower yields of hydroxyproline in Q - t r e a t e d tubes subsequently treated with the highest amounts of H20 2 may be explained, e.g., by assuming a shortening of the average free-radical chain-length due to presence of electron-scavenging molecules in these tubes. The evidence presented warrants the conclusion that 02 hydroxylates proline in the Udenfriend-Chvapil system in a manner that does not involve production of H~O 2 or of OH'. The relative insensitivity of the O 2 reaction to free radical inhibitors, arid to high iron concentration, makes it unlikely that free-radical species are involved in hydroxylation by O~. The mechanism for O 2 hydroxylation may be similar to one put forward by HAMILTON, WORKMAN AND WOO17 for O 2 oxidation of cyclohexane.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. (U.S.A.) I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17


H. S. MASON, Advan. Enzymol., X I X (1957) 79. R. BRESLOW AND L. N. LUKENS, J . Biol. Chem., 235 (196o) 292. J. H. GREEN, B. J. RALPH AND P. J. SCHOFIt~LD, Nature, 198 (1963) 754. S. UDEN~'RIEND, C. T. CLARK, J. AXELROD AND B. B. BRODIE, f . Biol. Chem., 208 (i954) 731. B. B. BRODIE, J. A~'~ELROD, P. A. SHOR~ AND S. UDENFRI~ND, J. Biol. Chem., 208 (1954) 741. M. CHVAPIL AND J. HURYCH, Nature, 184 (1959) 1145. D. FUJIMOTO AND H. TAMIYA, Biochem. dr., 84 (1962) 333. D. PROCKOP, i . KAPLAN AND S. UDENFRIEND, Biochem. Biophys. Res. Commun., 9 (1962) 162. YV. VAN B. ROBI~R:rSON AND B. SCHWAR~rZ, J. Biol. Chem., 2Ol (1953) 689. /3. S. GOULD, J. Biol. Chem., 232 (1958) 637. N. STONB AND A. MEISTER, Nature, 194 (1962) 555. B. PETBRKOFSKY AND S. UD~NFRIEND, Proe. Natl. A6ad. Sc*. U.S., 53 (I965) 335. W. VAN B. ROBERTSON, Connective Tissue: Intracellular Macromolecules, N e w Y o r k H e a r t Assoc. Syrup., Little, Brown, Boston, Mass., 1964. M. CHVAPIL, J. HURYCH, B. CMUCHALOVAAND 2 . EHRILICHOVA,in PH. COMTE, Syrup. Intern. : Biochimie et Physiologie du Tissu Conjontif, L y o n s , France, 1966, p. 455. J. F. WOESSNER, Arch. Biochem. Biophys., 93 (1961) 44 o. N. URI, Chem. Rev., 5 ° (1952) 375. G. A. HAMILTON, R. J. WORKMAN AND L. W o o , J. -//m. Chem. Soc., 86 (I964) 3390.

Received October 24th, 1967 Revised manuscript received December 22nd, I967 Biochim. Biophys. Acta, 156 (I968) 425-428