the metabolism of hydroxyproline

Volume 2, number 2 MOLECULAR & CELLULAR BIOCHEMISTRY 15 December 1973

THE METABOLISM OF HYDROXYPROLINE*

Elijah ADAMS

Department o f Biological Chemistry, University o f Maryland School o f Medicine, Baltimore, Maryland 21201

(Received August 10, 1973.)

Introduction

My interest in hydroxyproline began about twenty

years ago, while l was spending a training period in

biochemistry in the laboratory of Emil SMITH, then at

the University of Utah. Hydroxyproline impinged on

studies I undertook concerning the enzymatic hydrolysis of dipeptides such as X-proline (or

X-hydroxyproline) and prolyl-X (or hydroxyprolyl-X) 4 - 6. Additionally, I was influenced by Robert

NEUMAN (also a postdoctoral of SMITH'S at that time),

who was synthesizing a variety of proline and

hydroxyproline peptides as peptidase substrates. NEUMAN had recently completed doctoral work with

LOGAN at Cincinnati (work that led to the

development of a widely-used colorimetric method for hydroxyproline7), and publicized the subject of

hydroxyproline and collagen in our laboratory group. This interest was not immediately expressed in

research. After leaving Utah, I began independent

work at the National Institutes of Health, and for

some time studied the reactions and enzyme converting histidinol to histidine 8-10. Only after that work was

essentially completed, did I begin investigating the

metabolism of hydroxyproline. At that time (1954), whole animal studies with

i 5N_hydroxyprolinel i and 14C-hydroxyproline 12 had

indicated that hydroxyproline was oxidized at a substantial rate, and suggested the inference that

* This is a personal and informal account. More systematic reviews covering much of this material may be found in Refs 1-3.

glutamate was a major product m. Early cell-flee studies a 3, 14 suggested a mitochondrial oxidative

reaction like that of proline oxidase 13 as an initial

step. There was no substantial information concerning later steps.

Bacterial Studies

Overall Pathway Although identification of the products of

hydroxyproline metabolism in animals seemed

attractive because of its relation to collagen turnover,

my studies began with the isolation of a soil

microorganism, by the elective culture method, that

could grow on hydroxyproline as a sole source of

carbon and nitrogen.** From a patch of garden soil at the NIH, I isolated

two bacterial strains that grew well on hydroxy-L-

proline as the sole organic addition to media. One of

** This circuitous route to a study of hydroxyproline metabolism in animals was influenced by my introduction to elective culture methods by Dr. H. A. BARKER (at that time a visiting scientist at the NIH) and by the then current enthusiasm for the use of bacteria as tools in elucidating metabolic pathways that might be common to all cells. Successful examples of such studies then in progress at the NIH were RAB1NOWITZ'S explorations of purine degradation in Clostridia (summarized in (15)), a pathway that reversed some of the steps in the ubiquitous reactions of purine synthesis; and the studies by TABOR and associates of the degradation of histidine by a pathway essentially common both to Pseudomonas and animal cells (summarized in (16)).

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands 109

THE METABOLISM OF HYDROXYPROLINE

H

~N~N~ OOH

H

H

~ O O H

4-hydroxy-L -pro l ine 4-ollohyd roxy-D- prolin e

HO 4 3

N COOH H

HO OH

OOH

4-ollohydroxy-L-proline 4-hydroxy-D-proline

Fig. 1. Stereoisomers of 4-hydroxyproline. Ring positions are numbered as shown. The isomers of 3-hydroxyproline correspond to those shown for 4-hydroxyproline.

these strains proved to be a Pseudomonas* and has been maintained in our laboratory since that time. Whole-cell studies of this strain is showed that any of the four isomers of 4-hydroxyproline (Fig. 1) could be oxidized rapidly by cells induced with hydroxy-L- proline or allohydroxy-o-proline. Initial studies is with cell extracts suggested that L-glutamate was formed at an equal rate from either hydroxy-L-proline or allohydroxy-D-proline (Fig. 1). This was soon explained by the existence, in extracts of induced bacteria, of a highly active epimerase that rapidly equilibrated both epimers by racemizing carbon 2 (Fig. 2, Step 1). In the same report is, a particulate oxidase that converted allohydroxy-o-proline to the cyclic ketimine, Al-pyrroline-4-hydroxy-2-carboxylate (Fig. 2, Step 2), was also recognized. What proved later to be the remaining two steps of the pathway were correctly postulated on the basis of early fragmentary evidence: the deamination of the pyrroline intermediate to yield c~-ketoglutaric semial-

* Initially this strain was identified as Pseudomonas striata (18). Dr. R. Y. STANIER, using recent criteria published by his laboratory (17), has identified our strain as Pseudomonas putida, biotype A. It is maintained by the American Type Culture Collection (ATCC 15070).

[••COOH H

Pyrrole - 2- carboxylic acid

HO HO HO. 5/-H20 • , , . , .... I . " - 2

coT coo,O coo. Hydroxy-L-proline AIIohydroxy- D-praline AI-- Pyrroline-4 - hydroxy -

2- carboxylic acid

NH

COOH CHO C, H2 4 C, H2 CH2 -- CHz ,C=O TPN C,=O COOH COOH

'~ - Ketoglutaric ,x-Ketoglutaric acid semialdehyde

Fig. 2. Steps in the induced bacterial oxidation of 4-hydroxy-L-proline by Pseudomonas. Enzyme 1 is hydroxyproline-2-epimerase; 2, allohydroxy-o-proline oxidase; 3, Al-pyrroline-4-hydroxy-2-carboxylate deaminase; 4, a-ketoglutaric semialdehyde dehydrogenase. Reaction 5 is not known to be enzymatic but occurs spontaneously and is acid-catalyzed.

dehyde (Fig. 2, Step 3) and the dehydrogenation of the latter (Fig. 2, Step 4) to c~-ketoglutarate.

Most of our subsequent studies of this pathway have focused on the enzymes catalyzing individual steps, and these will be reviewed in the order of the pathway itself.

Hydroxyproline-2-epimerase This enzyme, induced 200-fold by exposure of cells to hydroxyproline, was purified to homogeneity about 10 years ago 19. This initial study provided the first amino-acid racemizing enzyme available as a pure protein. It was therefore of interest to investigate the enzyme for its content and utilization of pyridoxal phosphate, which had been implicated as a racemase coenzyme both by nonenzymatic-model studies and by kinetic studies of racemases (reviewed in 20). Our findings showed that hydroxyproline epimerase contained neither pyridoxal nor other plausible coenzymes 19, 2 a. A subsequent study of proline racemase by CARDINALE and ABELES led to a similar conclusion 22. In contrast, our strain of Pseudomonas contains an inducible alanine racemase which is a pyridoxal phosphate enzyme 23. The generalization has

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been proposed 2 that enzyme reactions involving pyridoxal phosphate are limited to the primary amino acids. In the case of proline and hydroxyproline, such reactions either do not occur (e.g., transaminases, decarboxylases) or where they do (e.g., racemases) they utilize alternative non-pyridoxal mechanisms to form the expected product.

A study of the mechanism of hydroxyproline-2- epimerase has engaged us since its purification. The initial finding that one of the substrate's hydrogens is exchanged with water during the reaction 19 led to a closer study by Thomas FINLAY 24, who showed that, as expected, this was the c~-hydrogen of hydroxyproline. Less predictably, however, its loss or gain (followed by N M R measurement of the incorporation or removal of deuterium) occurred at a rate parallel to the initial rate ofepimerization, suggesting a two-site mechanism 25. FINLAY'S subsequent observation 24 that two cysteines of the enzyme could be protected by substrate from inactivation by Ellman's reagent suggested that these two residues might represent the two sites implied by hydrogen-exchange data, and led to the formulation of steps shown in Fig. 3.

More direct evidence bearing on such a model is, of course, necessary. Constantine ZERVOS, in our laboratory, has shown that under specified conditions titration of the native enzyme with iodoacetate carboxymethylated one cysteine residue (of the twelve in the enzyme), with concomitant loss of enzyme activity. This single equivalent of cysteine appears to be divided between two tryptic peptides, separable by the fingerprinting technique. Preliminary indications were that the two peptides were clearly distinct in that one contained lysine and the other arginine 26,27, but their composition is now being thoroughly examined with larger amounts of material. Should the peptides prove to differ, this case would appear similar to that of ribonuclease, in which a single equivalent of iodoacetate alkylates either of the two active-site histidines 2 s, 29. The goal of sequencing

each peptide, in an effort to establish their identity or difference, has been thwarted by resistance of each to aminoterminal derivatization. A separate study of the S-carboxymethyl cysteines in each peptide has suggested that these were converted to the sulfones, and model studies of S-carboxymethyl cysteine sulfone indicate that this compound is resistant to dansylation, suggesting that in our active-site peptide(s), the cysteine is probably N-terminal.

-.,." coo- ,/2

cooo-

~' - , , ~ H o 0 - i

H( .H S/ &

H,z

I Jk . . . . o " ~ J . ~

~ . . - d ~ - DzOj7 ~ '

nv H:~ ~.~. ~ - -x,4#"

X/-~..CO0_ + " ~ ' ~ : ; " ' -s H

7 . . . . . ~ . ~ . ~ : u20 ~ ~I( ~ ~7

Fig. 3. Postulated steps in the hydroxyproline epimerase reaction. The active center is depicted as containing two sulfhydryls, one shown as deuterated. One epimer of 4-hydroxyproline (upper left) is pictured as binding to the enzyme through its amino, carboxyl and hydroxyl groups, and forming a carbanion intermediate which is converted to the other epimer by transfer of a hydrogen (deuterium shown here) from one sulfhydryl while the other accepts the former a-hydrogen of the substrate. This model was proposed to account for experiments with deuterium labeling and kinetic evidence for two active-site sulfhydryls (24); more recent studies (26, 27) have further supported it by the isolation of two dislinct peptides each containing an active-site sulfhydryl.

Allohydroxy-D-proline Oxidase Curiously, the oxidative metabolism of 4-hydroxyproline by Pseudomonas requires the preliminary epimerization of the most abundant isomer (that found in animals and higher plants) to one which is of only rare occurrence in nature. This was demonstrated most dearly by the finding that an epimerase-lacking

11I


mutant is unable to utilize hydroxy-L-proline for growth or for any appreciable conversion to other compounds 3°. The D-epimer, produced normally by the epimerase, is, however, the substrate for an inducible, particulate enzyme which carries out a dehydrogenation analogous to that of other D-amino acid oxidases, yielding the cyclic ketimine (Fig. 2). We have learned relatively little about this step because of its enzymatic complexity, involving a sequence of electron-transfer reactions to cytochromes 3a. An earlier effort to solubilize the primary dehydrogenase seemed successful both from the ultracentrifugal behavior of the "solubilized" enzyme and its loss of reactivity with oxygen, except in the presence of phenazine methosulfate 32. Later studies, based on gel filtration, indicated that, while not sedimentable at high speeds, the enzyme was still associated with a large unit and was still undoubtedly complex. Its apparent large size (1-2 million), despite apparent "solubility" by centrifugation criteria, suggests significant lipid content, verified by preliminary measurement (E. ADAMS and M. D. MURRAY, unpublished).

A a-Pyrroline-4-hydroxy-2-carboxylate Deaminase The development of a method 33 for large scale enzymatic preparation and isolation of the pyrroline intermediate, a rather unstable compound, permitted definition of the next enzymatic step. This proved to be a non-oxidative ring-opening reaction accompanied by deamination (Fig. 2, Step 3). The deaminase was

,o COOH H2N 0 COOH H2N 0 COOH

A I- Pyrroline-4-hydroxy- 2-carboxylate lr

H20

-NH 3 0 OOH HN COOH

,x-Ketoglutaric semialdehyde

Fig. 4. Possible intermediate steps in the conversion of Al-pyrroline-4-hydroxy-2-carboxylate to a-ketoglutarate semialdehyde. The pyrroline substrate can be viewed as a potential equilibrium partner of the open chain a-keto acid; the latter compound, having vicinal hydroxyl and amino groups, could undergo reactions analogous to those of serine or threonine dehydration with subsequent loss of the amino group as NH3.

purified to apparent near-homogeneity33; no cofactor was implicated by testing several types of possible inhibitors. While the mechanism of the reaction has not been studied, a reasonable analogy appears to be that of the serine-threonine dehydrase reaction (Fig. 4). Although the latter group of enzymes utilize pyridoxal phosphate, inhibitor studies of the pyrroline deaminase cited above gave no indication of a pyridoxal cofactor.

c~-Ketoglutarie Semialdehyde Dehydrogenase The product of the preceding enzyme, ~-ketoglutaric semialdehyde (or 2,5-dioxovalerate), was first proposed as a hypothetical intermediate in proline-glutamate interconversions 34. As a demonstrated metabolic intermediate, it first came to light in the present studies33, 35, 36, but its involvement in several other

bacterial pathways leading from pentoses or hexarates to c¢-ketoglutarate was soon after reported 37- 39.

Aware that the enzymology of glutamate reduction to form A~-pyrroline-5-carboxylate is still unclear, we considered seriously KREBS' early suggestion 34 that ~-ketoglutaric semialdehyde (KGSA) might be such an intermediate; however, our studies failed to support the possibility that unlabeled ~-ketoglutaric semialdehyde might be a direct proline precursor in rats or rat tissues, or that it might be formed from a-ketoglutarate in animal tissues or bacteria 4°.

The availability of ~-ketoglutaric semialdehyde as a substrate made it possible to undertake a study of the final step of hydroxyproline catabolism in bacteria (Fig. 2, Step 4), the TPN-linked oxidation of c~-ketoglutaric semialdehyde to ~-ketoglutarate. An investigation of this enzyme in our Pseudomonas strain41, ,~2 soon showed an apparent difference from the three preceding enzymes of this pathway. In contrast to the 50- to 200-fold increase in each of these enzymes by induction 3°, KGSA dehydrogenase was increased only about 10-fold by growth of cells in the presence of hydroxyproline. The relatively high "basal" level of the enzyme proved to represent a distinct protein whose difference from the induced enzyme in molecular weight, electrophoretic behavior and substrate specificity was readily demonstrable 4l ' 42

The "basal" isoenzyme proved, in studies not yet completely reported 43, to be induced by lysine and to represent a step in the catabolism of L-lysine in Pseudomonas (Fig. 5); it bad previously been recognized and partly purified in this connection 44. Efforts to clarify the isoenzyme status of the lysine-

112


CH2NH 2 CH2NH 2 CH2NH 2 CHO COOH (CH2)5 02 (CH2)5 -NHs,,, (C, H2) 3 ~ (CH2)3 TPN (iH2)3

H COOH cNH2 o~C "" NH2 COOH COOH COOH

L- Lysine &-Aminovaleramide ~ -Aminovolerate Glutarate Glutarate semialdehyde

Fig. 5. Reactions in the induced catabolism of L-lysine in Pseudomonas. The enzyme catalyzing the last step shown (glutarate semialdehyde dehydrogenase) also acts on a-ketoglutarate semialdehyde and was first recognized in our strain as the apparent "basal" level of a-ketoglutarate semialdehyde dehydrogenase.

induced dehydrogenase in our strain led us into a rather extensive investigation of lysine metabol ism in this strain, published only in part es.

While we were purifying and characterizing the hydroxyproline-induced K G S A dehydrogenase 42,

other studies indicated that in certain Pseudomonas

strains, glucarate or galactarate also induced a K G S A dehydrogenase 37, (Fig. 6) as does growth (in some

strains) on arabinose 38 (Fig. 7). We soon learned that

our strain also grew well on glucarate as a carbon source, but apparently did not so utilize arabinose.

After growth on glucarate, just as after growth on

hydroxyproline, our cells contained high levels of

K G S A dehydrogenase, and it appeared of interest to

learn if the glucarate-induced and the hydroxyproline-

induced enzymes were identical or not. On an a priori

basis, either result would have precedent. Separately- induced and distinct catabolic pathways in bacteria

which converge on a common intermediate (as was the

COOH H ,C-OH

HO-,C-H H-C-OH H-,C-O H " ~ COOH

COOH H C'-OH D- Glucarate HO-C,-H

CH 2

COOH _~H2 0 C:O H (;:-OH COOH HO-C-H H 0 -(~ - H D-4- Deoxy-5- H',C-OH oxocjlucarate

COOH

D- Galactarate

_H2 0 HC:O COOH CH 2 DPN + ¢H2

-CO~ ,CH2 ,- CH2 C=O C=O ~OOH ~OOH

w-Ketoglutarate .~-Ketoglutarate semialdehyde

Fig. 6. Reactions in the induced catabolism of D-glucarate and D-galactarate in Pseudomonas. A step common to both hydroxyproline and saccharate metabolism is the oxidation of ct-ketoglutarate semialdehyde to ct-ketoglutarate. Enzymes catalyzing the intermediate steps have been described by JEFFCOAT et al (88, 89).

COOH COOH COOH COOH i i i i

H-C-OH %0 c:o, _H2 o C:O, DPN + C--O, HO-C-H ,- ,(;H 2 ,- C,H 2 ,,-,CH z

i

HO-C-H HO-C-H CH 2 ,CH 2 i i

H2C-OH H 2 C-OH CHO CliO

L-Arabonate L-2-Keto-.3- deoxy ,t- Ketoglutarate ,~-Ketoglutarate arabonate Semialdehyde

Fig. 7. Reactions in the conversion of L-arabonate to a-ketoglutarate via a-ketoglutarate semialdehyde (38). Our strain is unable to grow on arabinose as a carbon source.

case here) have been shown to involve two distinct

enzymes for the first convergent step, each enzyme

induced by the corresponding inducer for each individual pathway ¢6' 47. On the other hand, one can

find examples of such a step, c o m m o n to two or more

pathways, which is catalyzed by a single enzyme,

induced either by the common substrate or a later

product ¢8. In our initial studies, the K G S A

dehydrogenase induced by hydroxyproline was not readily distinguished f rom that induced by hydroxy-

proline 4z, and suggested identity of the two enzymes,

implying a common inducer. However, the logical

Table 1

Differences between Hydroxyproline- and Glucarate-induced KGSA Dehydrogenase

Criterion Comment

Electrophoresis

Peptide structure

Immunological

Genetic

Separate protein bands on polyacrylamide gels, either singly or after co-induction of both enzymes in the medium Maps of tryptic peptides of each enzyme show no common peptides Antisera to each enzyme give separate bands by immunoelectrophoresis or immunodiffusion. Some cross-reactivity by enzyme inactivation A structural mutation in the hydroxyproline-induced enzyme and a regulatory mutation preventing induction of the entire hydroxyproline pathway permit normal induction of the glucarate-induced enzyme

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Table 2

Similarities in the Hydroxyproline- and Glucarate-induced KGSA Dehydrogenase

Criterion Comment

composition, in the case of each enzyme. A literal interpretation of the data so far at hand would predict extensive sequence replication in each protein to account for these results; however a more searching examination of other possible interpretations is called for.

Molecular Weight Subunits

Amino Acid Composition

N-terminus Kinetic Data

Substrate Specificity

Other enzymatic activity

Both about 120,000 Two, apparently identical, in each enzyme Similar, Metzger index 8.551 Arginine Km for KGSA, glutarate semialdehyde, DPN, TPN, not distinguishable; Vmax ratios for these substrates not distinguishable Other aldehyde substrates show relative substrate activity or inhibition of KGSA, not distinguishable for the two enzymes. Both contain low activity of A 1-pyrroline-4-hydroxy- 2-carboxylate deaminase

1 See (90).

candidates, KGSA itself, or c~-ketoglutarate, were later shown to be devoid of inducer activity for this enzyme; and instead hydroxy-L-proline was an inducer 3°. It therefore appeared that if the separately-induced enzymes were indeed identical, this might imply a common gene regulated by two otherwise independent induction systems, a case which we believed had not been documented in patterns of catabolic regulation.

To answer this question, Peter Koo, then a graduate student, undertook to purify "both" enzymes to homogeneity, preparatory to their detailed comparison both as enzymes and proteins. This was accomplished, and on genetic, immunological, and structural grounds, the conclusion was clear that the two enzymes were different (Table I) despite marked similarities including kinetic similarity with respect to Km's, and relative activity with a number of aldehyde substrates (Table II) 49.

An unresolved question which this study raised was a markedly lower number of tryptic peptides than that expected from subunit size and amino acid

Transport and Regulation A general investigation, by Dr. Rosa GRYDER 30, of induction properties indicated block induction of the entire pathway by hydroxy-L-proline: the clearest evidence for this came from a mutant strain lacking hydroxyproline epimerase, in which no conversion of the inducing substrate to the later intermediates occurred. It appeared (as is true for certain other bacterial pathways, notably that of the lac operon) that hydroxyproline transport is similarly induced, and that a regulatory mutant lacking the capacity to induce any of the pathway enzymes is also unable to carry out hydroxyproline transport s°,

The availability of an epimerase-negative mutant, in which hydroxy-L-proline is metabolically isolated, made it attractive to characterize some features of this inducible transport system s°. Kinetic data led to the formulation

VS~ v -- - - keS i

Km@S e

in which, v, the net velocity of hydroxy-L-proline uptake, is determined by a saturable entry process characterized by an uptake maximal velocity, V, and an apparent K m for the external hydroxyproline concentration, S e. The uptake rate is balanced by an exit rate, related through a first-order rate constant, k~, to Si, the internal hydroxy-L-proline concentration. In fully-induced cells, values for K m and V were calculated respectively to be 3 × 10- s N and 6/~moles per g dry cell weight. The value of V increased 3- to 4-fold with induction by hydroxy-L-proline; the exit process (k e = 0.23 m i n - 2) appeared non-inducible.

In general features, the uptake system is analogous to that for other energy substrates, such as galactose 51, s2, rather than to those for amino acids. It is

notable that the uptake system for hydroxy-L-proline is much more efficient than for allohydroxy-D-proline (Km ,-~ 10 -3 M, Vma x ~' 0.1 /~mole per g dry cell weight), even though the L-epimer can only be utilized via preparatory conversion to the D-epimer.

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Animal Studies

Unlike bacteria, animals and higher plants contain appreciable hydroxyproline, almost entirely present in structural proteins. The metabolism of the free amino acid by these organisms therefore has a different level of physiological relevance than in bacteria, where such reactions can be viewed primarily as an example of energy opportunistically procured from a substrate in the soil.

As has been discussed 2, the significance of a degradative pathway for hydroxyproline in animal ceils is not clear. Hydroxyproline produces no intermediates of known physiological utility, its energy contribution is quantitatively insignificant, and it does

CHO HC-OH ,CH2

HC-NHz COOH

4- Hydroxygluto mic I semioldehyde

HO.... ".. H~,-OH

HC,-,H a COOH COOH COOH

H A 1- Pyrroline-3-hydroxy- 4-erythro-Hydroxy-L- Hydroxy-LLprol ine 5-carboxylic acid glutamic acid

COOH CHO COOH

Glyoxylic acid 5 H~-OH + C, H2

,CH3 H,C=O ,C=O COOH COOH 4- Hydroxy-

Pyruvic acid 2- keloglutaric acid

Fig. 8. Steps in the major pathway of hydroxy-L-proline in mammals. Enzyme 1 is the particulate hydroxyproline oxidase which appears to differ from mitochondrial praline oxidase (see text). Enzyme 2 is a DPNH-linked reductase for Al-pyrroline-3-hydroxy-5-carboxylate. Enzyme 3 is a DPN-linked dehydrogenase. Enzyme 4 has not been distinguished from glutamic-aspartic transaminase. Enzyme 5 is 4-hydroxy-2-ketoglutarate aldolase. Reaction 6 is not known to be enzyme-catalyzed and is not written as a step between Al-pyrroline-3-hydroxy-5-carboxylate and 4-hydroxyglutamate for reasons presented elsewhere (61). Not shown is a minor reaction representing the oxidation of hydroxy-L-proline by kidney L-amino acid oxidase and yielding Al-pyrroline-4-hydroxy-2-carboxylate (see Fig. 2), evidence for which is presented in (83).

not appear to be so toxic that its removal is warranted for this reason. Nevertheless, our studies have defined a rather active pathway for free hydroxy-L-proline in animal tissues, capable in man of oxidizing almost completely the 250 mg (or more) of free hydroxy- praline arising daily by collagen turnover 53' 54, and

with a considerably greater potential capacity tested by the administration of large quantities of exogenous hydroxyproline 2. This pathway as presently understood is outlined in Fig. 8, and will be discussed in more detail in the order of individual reactions.

Hydroxy-L-proline Oxidase and the Pyrroline Product The first reaction produces a cyclic aldimine, a A 1- pyrroline (different from that in the bacterial pathway) in which two hydrogens have been removed from nitrogen and carbon 5 (Fig. 8, Step 1). As noted earlier, this reaction was postulated in early studies 1 a, 14, although the product was not obtained as such and hence was not available as a substrate to explore subsequent reactions. Our own studies of the animal pathway began with a large scale isolation of A 1- pyrroline-3-hydroxy-5-carboxylate and its partial characterization 55. For this, a crude preparation of the oxidase in kidney mitochondria was utilized.

The enzyme itself has not been definitively studied, because of difficulty in its solubilization*. For the same reason, biochemical separation of hydroxyproline oxidase from praline oxidase has been reported only recently in preliminary form 57. However, the existence of distinct clinical entities seemingly involving a genetic absence of hydroxyproline oxidase 53' 54 or praline oxidase 58, implies the distinctness of these two enzymes.

Reduction and Oxidation o f A1-Pyrroline-3-hydroxy-5 - carboxylate Both a soluble reductase and a soluble dehydrogenase which act on the pyrroline substrate are present in mammalian liver (Fig. 8, Steps 2, 3). The product of the reductase reaction is hydroxy-L-prolineS9; its distinctness from the enzyme which reduces A 1- pyrroline-5-carboxylate to L-praline 6° has not been

* Brief reports by KRAMAR have described the solubilization of praline oxidase, first by detergents (56) and subsequently by snake venom (57); hydroxyproline oxidase, however, was selectively inactivated by the latter solubilization procedure, and this observation reinforces the belief that the two reactions involve separate enzymes.

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demonstrated. The role of this reaction in metabolism is unclear. If the enzyme should prove to be distinct from the analogous enzyme which serves to synthesize proline, it would imply some function whose meaning is quite obscure, since no route to Al-pyrroline 3-hydroxy-5-carboxylate is known except from hydroxy-L-proline itself. Alternatively, the reduction of the hydroxy-analog may represent only a side action of an enzyme whose physiological role is the formation of proline. It is of interest, however, that Al-pyrroline-5-carboxylate reductase from Neurospora, an organism not known to contain hydroxyproline, failed to catalyze the analogous reduction of A 1- pyrroline_3.hydroxy_5_carboxylate 59.

The dehydrogenase that acts on the pyrroline product of hydroxyproline oxidase (Fig. 8, Step 4) has also been partly purified from rat liver 61. Its relation- ship as a protein to the enzyme catalyzing an analogous oxidation of Al-pyrroline-5-carboxylate to L-glutamate is similarly unresolved 61' 62. The product

of the hydroxypyrroline substrate was shown to be erythro_4_hydroxy_L_glutamate 61, providing additional validation to the steric identification of the isomers of 4-hydroxyglutamate. This intermediate, which had been suggested by fragmentary findings in other laboratories63, 64, was isolated as an enzymatic product and definitively characterized 61. Its only earlier recognition in nature was as a plant product 6s.

Further Reactions of 4-Hydroxyglutamate Further studies of the metabolism of 4-hydroxy-L- glutamate were carried out in several laboratories. The first suggestion concerning its fate was that of direct cleavage to yield glyoxylate and alanine66; other reports67, 6s indicated that this overall reaction was

probably the sum of several steps: transamination of 4-hydroxyglutamate to yield 4-hydroxy-2-ketoglutarate, with subsequent cleavage of the latter keto acid to yield glyoxylate and pyruvate; alanine then accumulated as a transaminase product of pyruvate.

The transamination reaction of 4-hydroxyglutamate appeared to be catalyzed by glutamate-aspartate transaminase of rat liver 6s and has not been further studied as a reaction characteristic of hydroxyproline metabolism. The cleavage of hydroxy-ketoglutarate, however, has been extensively studied both by our- selves69 - 71, and by DEKKER and his associates 7z- 75,

particularly since it offered points of novel difference from other aldolases. Thus it was shown by us and

subsequently confirmed by DEKKER'S laboratory that the aldolase was sterically non-discriminating 6 s, 76, 7 a

and would catalyze, with essentially equal efficiency 7° the cleavage and synthesis of both L- and D-4-hydroxy- 2-ketoglutarate. Furthermore, the binding of both substrates, glyoxylate and pyruvate, as Schiff bases with a lysyl residue of the enzyme 69' 73, represented a difference from other aldolases in which the condensing fragment capable of so binding an enzyme lysine is restricted to that fragment with carbanion forming capacity in the aldol condensation. More recent studies of the purified enzyme, which was originally obtained as an essentially pure protein from rat liver 69 and beef liver 74, have been extended by Dekker's laboratory to a bacterial enzyme 7 s.

While the metabolism of 4-hydroxyglutamate via transamination to the corresponding ~-keto acid may well be a major route quantitatively, other biological reactions which 4-hydroxyglutamate can undergo have not been evaluated with respect to their quantitative aspects or the extent to which they may form physiologically active products in animals. We have ourselves reported the synthesis of 4-hydroxyglutamine from either erythro- or threo-4-hydroxy-t,-glutamate by mammalian enzymes 77' 7s, and the decarboxylation

of the mixed isomers of 4-hydroxyglutamate by rat brain has been reported 79. The formation of both threo- and erythro-isomers of 4-hydroxyglutamate might be anticipated in vivo, since both the transaminase and aldolase (which catalyze the reversible conversion of 4-hydroxyglutamate to glyoxylate and pyruvate) are sterically non-selective for the configur- ation of the hydroxyl group. Through this route alone, it is thus possible to anticipate a variety of labeled products (at least in trace amounts) from the metabolism of hydroxyproline in animals.

Related Studies

3-Hydroxyproline This position isomer, in which the hydroxyl group occupies position 3 of the pyrrolidine ring (Fig. 1), was first discovered as a minor component of verte- brate interstitial collagen 8°. It has assumed wider interest because of its relatively high concentration in basement-membrane collagen s 1, but little is known of its metabolism either in animal tissues or bacteria. We have surveyed initial reactions of 3-hydroxyproline

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both by bacterial and animal enzymes of known defined activity for specific epimers of 4-hydroxyproline s2. It is of interest, but has not yet been further investigated, that 3-hydroxyproline induces the utilization of 4-hydroxyproline by Pseudomonas3°; whether it will be found that a common pathway exists (which seems unlikely on structural grounds), or that this only represents analog inducer activity, will require further study.

Minor Pathway of 4-Hydroxyproline In a current study of the source of pyrrole-2-carboxylate excreted in mammalian urine as, Anne HEACOCK, a student in our laboratory, has shown that hydroxy-L-proline is a precursor of the pyrrole. Since no mammalian pathway was known that could account for this direct conversion, it was earlier considered 2 that the bacterial flora of the intestine might furnish a crucial reaction, namely the epimerization of hydroxy-L-proline to allohydroxy-D-proline. This possibility, however, appears unlikely from HEACOCK'S experiments, in which rats whose gastrointestinal contents were rendered almost sterile by antibiotic treatment showed no reduction in their capacity to excrete the pyrrole after a hydroxyproline load. A plausible enzymatic source for this conversion was found in the kidney oxidase which oxidizes L-hydroxy- or L-amino acids to the corresponding keto acids 84. This enzyme, whose capacity to act on hydroxy-L-proline had not been reported, catalyzes a slow oxidation of this substrate yielding the cyclic ketimine which readily loses water to form pyrrole-2- carboxylate (Fig. 2). The possible usefulness of this excretion product as an index of collagen breakdown seems worth further study, since it may yield information beyond that obtained from measurements of hydroxyproline excretion itself 85.

Several features of pyrrole carboxylate formation and excretion remain unexplained. Among these are the findings that in rats a proline load also increases excretion of the pyrrole and the requirement of rather large hydroxyproline loads to increase appreciably the low level of endogenous pyrrole-2-carboxylate excretion. Thus, although present data indicate free hydroxy-L-proline as the source of the pyrrole via the enzymatic route noted 83, a number of puzzling questions require clarification before this picture is fully validated.

Future Problems

A subject on which there is little detailed knowledge concerns the origin of free hydroxyproline in animals. Essentially by default, - - i.e., through lack of plausible alternative routes, some of which have been considered and examined 2, - - it is believed that free hydroxyproline originates entirely from the breakdown of collagen or collagen-like sequences, such as those in the complement component C-lq a6.

A question of interest concerns the extent to which hydroxyproline is released from mature collagen molecules relative to its release from newly-made collagen. There are data to suggest that collagen synthesis is associated with the release of low-molecular weight peptides containing hydroxyproline (reviewed in (2,87)); an explanation for this association has not been established.

An additional investigative area of interest concerns the metabolism of 3-hydroxyproline, the possibility that it may yield unique metabolic intermediates or products and the extent to which turnover of basement membrane collagen may be reflected by the release of 3-hydroxyproline and/or the production of its metabolites.

Acknowledgement

The studies summarized in this review were supported by consecutive grants from both the National Science Foundation (Grants G-11419, B-4577, G-5207) and U.S. Public Health Service (Grants E-2444, GM-07192, and GM-11105), dating from 1956 to the present.

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the metabolism of hydroxyproline

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