[105] Histidine degradation (formiminoglutamic acid metabolism)

784

[105]

ENZYMES OF PROTEIN METABOLISM

[ 105] Histidine Degradation (Formiminoglutamic Acid Metabolism) 1 By

CH + I H HOOC-CH-CH2CHzCOOH .FORMIMINOG~TAMIC. A~ID

HERBERT TABOR

TRANSF'ERASI[j ~1

N~4,,.N~ .CHz.NH_R OH H

-~.~E~QW~¢ ACID (,THF)

~.~.~

H

H

.ooo-o..o..~..~oo.

~

(,

I NH GLUTAMIC ACID

H

5- FORMIMINO-THF

FORMIMINO-THF

H

N-R

OR

" " N HCdH -R',.m'I

H+

,

"P NH3

(~

OH N I ~ c H ~

NH 5- FORMIMINO-THF

"."r~",~" 1

.

..o

= °

.5m10- METHI[NYL-THF

(:,)

.÷ IO-FORMY~-THF

1The procedures presented here have been reported by H. Tabor and L. Wyngarden, Y. G/~. l~tvest.. 37, 824 (1958) and J. Biol. Chem. 234, 1830 (1959). These

T I. Formimino-L-glutamic Acid F o r m i m i n o - T r a n s f e r a s e

Assay Method Principle. The assays for both this enzyme and cyelodeaminase are based on the high extinction coefficient (24,900) of 5,10-methenyltetrahydrofolic acid at 350 m~ in acid and at 358 m~ at p H 7. I n the assay for the formimino-transferase, the enzyme preparation is incubated with excess formimino-L-glutamic acid and excess t e t r a h y drofolic acid; formiminotetrahydrofolic acid is formed. Perchloric acid is then added; this both precipitates the protein and converts the formiminotetrahydrofolic acid to 5,10-methenyltetrahydrofolic acid. The increase in optical density at 350 m~ is then measured to determine the amount of 5,10-methenyltetrahydrofolic acid formed. Reagents

1 M potassium phosphate buffer, p H 7.2. 0.1 M sodium formiminoglutamate. The preparation of this compound is described in Vol. VI. O.O07M sodium d/-tetrahydrofolate in 1 M mercaptoethanol. A preparation of tetrahydrofolic acid is described in Vol. VI. I m m e d i a t e l y after the synthesis, a stock solution of sodium tetrahydrofolate (0.07M) is made by adding the appropriate amount of 1 M mercaptoethanol to tetrahydrofolic acid, and adjusting the p H to approximately 7 with N a O H . This solution can be stored in the d a r k at 0 ° in v a c u a for at least 6 months. For use in the assay procedure, this stock solution is diluted 1:10 with 1 M mercaptoethanol and stored at 0 ° in vacuo. 3

articles contain references to previous publications by various investigators in this area. The formulations and assays presented here for the metabolism of formiminoglutamic acid by liver preparations are analogous to the reactions previously described by J. Rabinowitz and W. E. Pricer, Jr., for the metabolism of formiminoglycine by purified preparations from Clostridium cylindrosporum and Clostridium addi-udd (see Vol. VI). I T he preparation of cyclohydrolase from liver (J. Biol. Chem. 234, 1830, 1959) is not being presented in this section. Refer to the article by D. M. Greenberg (Vol. VI) for the preparation of liver cyclohydrolase. A cyclohydrolase from Clostridia extracts has been described by J. Rabinowitz and W. E. Pricer, Jr. (J. Am. Chem. ~Joe. 78, 4176, 1956).

' It is usually not necessary to ~say these solutions for their tetrahydrofolic acid content. When a determination of the tetrahydrofolic acid concentration is desired, a modification of the formimino-transferase assay can be used, in which excess formiminoglutamic acid and enzyme are incubated with limiting amounts of tetrahydrofolie acid. Further details are included in the publications cited in footnote 1 and in Vol. VI.

786

ENZYMES OF PROTEIN METABOLISM

[105]

10~ perchloric acid. Enzyme. Dilute the enzyme immediately before use with cold 0.5 M potassium phosphate buffer, pH 7.2; the diluted solution should have approximately 50 units/ml.

Procedure. To a 3-ml. test tube in a 25 ° water bath are added potassium phosphate buffer (0.1 ml.), sodium tetrahydrofolate (0.1 ml.), sodium formiminoglutamate (0.05 ml.), water (0.74 ml.), and diluted enzyme (10 ~). The solutions are rapidly mixed and incubated for exactly 2 minutes. Perchloric acid (0.3 ml.) is added immediately, and the solution is heated in a boiling water bath for 55 seconds. The solution is immediately cooled in ice water and centrifuged. The supernatant solution is transferred to a cuvette (1.2-ml. volume, 1-cm. light path), and the optical density is measured at 350 m/~. For the reference cuvette a control incubation mixture is used, which is treated in a similar manner, except for the omission of the sodium formiminoglutamate. Definition of Unit and Specific Activity. The number of units of enzyme in the aliquot used is calculated by multiplying the increase in optical density * per minute by the final volume (1.3 ml.). Specific activity is expressed as units per milligram of protein. Protein is determined by measuring the absorption at 280 m/~ and 260 m/~ (Vol. III [73], p. 451). Application o/ Assay Method to Crude Tissue Preparations. This assay is suitable for use with crude liver preparations. The presence of cyclodeaminase or o f cyclohydrolase does not affect the assay, since, on acidification, all the products are converted to 5,10-methenyltetrahydrofolic acid. Purification Procedure

S~ep 1. Acetone Powder Extract. Hog liver is used as the starting material; commercial lots of frozen hog liver are satisfactory and can be stored a t - - 2 0 ° for at least 6 months without significant loss in activity. Acetone powders are prepared essentially as described in Vol. I [6], p. 35, and Vol. I [101], p. 609; it is satisfactory to prepare these powders at room temperature. The acetone powders are stored at --15 ° in vacuo, with a silica gel drying agent, with essentially no loss in activity for as long a s 6 months. Acetone powder (320 g.) is extracted with 3200 ml. of water at room temperature for 15 minutes with occasional gentle stirring. The suspension is then centrifuged for 20 minutes at 20,000 X g, and the super~A change in optical density of 1.0 represents 0.04 micromole of 5,10-methenyltetrahydrofolic acid per milliliter, or 0.052 micromole in an assay volume of 1.3 ml.

[10S]

HISTIDINE DEGRADATION

787

natant fluid is collected. All centrifugations are carried out in an angle centrifuge. All subsequent steps are at 0 ° to 2 °, unless otherwise indicated. Step ~. Ammonium Sul]ate Fractionation. Ammonium sulfate (600 g.) is added to 3000 ml. of this supernatant solution. After approximately 2 hours this is centrifuged at 20,000 X g for 20 minutes, and the supernatant solution is discarded.

Step 3. Dif]erential Extraction o] the Ammonium Sulfate Precipitate. The precipitate is thoroughly suspended in 65 ml. of water and stored for about 18 hours with gentle magnetic stirring. The murky mixture is then centrifuged for 2 hours at 35,000 X g; the supernatant is discarded. The residue is extracted with approximately 75 ml. of 0.2 M sodium acetate with gentle magnetic stirring. After 4 to 18 hours the suspension is centrifuged at 35,000 X g for 30 minutes, and the supernatant is collected.5, 6 Step ~. pH Fractionation. To the above supernatant solution 0.1 vol. of a 3 M acetate buffer (sodium acetate:acetic acid, 90: 10) is added. After approximately 5 hours this is centrifuged for 30 minutes at 35,000 X g. The supernatant solution is discarded, and the precipitate is suspended in 15 ml. of 0.1 M triethanolamine sulfate buffer, pH 7.2. Step 5. Chymotrypsin Treatment. As noted in the table, the above steps do not separate the formimino-transferase from the cyclodeaminase. To accomplish this, the enzyme solution is incubated with chymotrypsin, since this results in a more rapid inactivation of the cyclodeaminase than of the formimino-transferase. The enzyme solution obtained in step 4 is brought to 25 ° and diluted with 1.5 vol. of water. For each milliliter of enzyme, 0.025 ml. (0.5 mg.) of a solution of chymotrypsin (Worthington Biochemical Corporation; "crystallized, salt-free") is added. After 2 hours at 25 °, the ehymotrypsin is inactivated by the addition of a 0.2 M solution of diisopropylfluorophosphate in isopropanol (0.007 ml./ml, of the enzymeehymotrypsin mixture). Sufficient 0.5 M triethanolamine sulfate buffer, pH 7.2 (0.12 ml./ml.), is then added to give a final concentration of 0.1 M buffer (preparation 5). This preparation can be stored at 2 ° for several weeks without loss ° The preparation at this stage is approximately 250-fold purified and is suitable for most studies, such as the assay of formiminoglutamic acid (J. Clin. Invest. 37, 824 (1958) and Vol. ¥I). These preparations can be stored at 0° for at least 3 months or at --20 ° for at least 6 months with no loss in activity. No cyclohydrolase activity is present in this preparation. i Additional enzyme activity can be obtained, if desired, by re-extraction of the pellet with 75 ml. of 02 M sodium acetate for 18 hours.

788

[105

ENZYMES OF PROTEIN METABOLISM

of activity, but it loses 16 to 45% of its activity after 2 months a t 2 ° At - - 2 0 ° some preparations can be stored without loss of activity, bu other preparations are v e r y unstable under these conditions. A s u m m a r y of the purification procedure is given in the table. PURIFICATION OF FORMIMINOGLUTAMIC ACID FORMIMINO-TRANSFERASE AND CYCLODEAMINASE FROM HOG LrVER ACETONE POWDER

Cyclodeaminase

Formimino4ransferase Step 1. Extract of acetone powder 3. NaAc extract of (NH4)2S04 precipitate 4. pH fraetionation

Volume, Protein, ml. mg.

3000 77 15.4

Total units

Specific activity, units/mg,

Total units

110,000

123,000

1

281,000

193 53

48,700 27,700

250 520

120,000 70,300

Specific activity, units/rag. 2.6 620 1330

SEPARATION OF FORMIMINo-TRANSFERASE FROM CYCLODEAMINASE BY TREATMENT WITH CHYMOTRYPSIN OR WITH AMMONIA

5. After ehymotrypsin treatment 5'. After ammonia treatment

46

20,600

25

18

4

16,600

Properties

Specificity. This preparation is relatively specific for formimino glutamic acid. No reaction is found when formiminoglycine 7 is use~ as a substrate, and only a v e r y small activity ( < 0 . 1 % of the rate wit] formiminoglutamic acid) when formimino-L-aspartic acid or formyl-L glutamic acid s is used. Equilibrium Constants; pH Optimum; Salt Effects. Reaction (1) i reversible with an equilibrium constant of 0.8 to 2.6. M a x i m u m activit: is observed between p H 7.1 and 8.1. A small stimulation is observed witl low concentrations of KC1 or NH~C1. A 0.1 M concentration of a variet~ of salts has an inhibiting effect. vSee J. Rabinowitz (Vol. VI) for a formiminoglyeine formimino-transferase fron Clos~ridla extracts. *See M. Silverman (Vol. V [106]) for the preparation frSm hog liver of al enzyme which transfers the formyl moiety from formyl-L-glutamic acid to tetra hydrofolic acid [M. Silverman, J. C. Keresztesy, G. J. Koval, and R. C. Gardinm Y. Biol. Chem. 226, 83 (1957)].

[105]

HISTIDINE DEGRADATION

789

Affinity Constants. The K~ for formimino-L-glutamic acid is 1.1 X 10-2 M. The K~ for l-tetrahydrofolic acid is 1 X 10-' M. II. Formiminotetrahydrofolic Acid Cyclodeaminase g

Assay Method Principle. The enzyme is incubated with formiminotetrahydrofolic acid. The increase in absorption at 358 m~ represents the amount of 5,10-methenyltetrahydrofolic acid formed. During most of the purifiication procedure, the cyclodeaminase arid formimino-transferase activities are not separated, and hence a separate assay for the eyclodeaminase is usually not necessary.

Reagents Potassium maleate buffer, pH 6.5; 1 M with respect to K ÷. 0.1 M mereaptoethanol. 0.002 M formiminotetrahydrofolic acid. This compound can be prepared with formiminoglutamic acid and the purified formiminotransferase described above (details are given by Tabor and Wyngardenl). The preparation of formiminotetrahydrofolic acid with formiminoglycine and a purified Clostridia formiminotransferase is described by J. Rabinowitz in Vol. VI. Enzyme. The enzyme is diluted immediately before use in the potassium maleate buffer to a concentration of approximately 50 units/ml.

Procedure. Potassium maleate buffer (0.2 ml.), formiminotetrahydrofolic acid (0.05 ml.), 0.1 M mereaptoethanol (0.02 ml.), water (0.72 ml.), and enzyme (0.01 ml.) are mixed in a cuvette (1-cm. light path; 1.2-ml. volume), and the increase in optical density is measured at 358 m~ in a recording spectrophotometer. The reference cell contains all the components except the enzyme. Definition of Unit. One unit of enzyme is defined as that amount of enzyme which causes an increase in optical density of 1.0 per minute under the above conditions2 Application o] Assay Method to Crude Tissue Preparations. This assay is not suitable for any preparation containing large amounts of eyclohydrolase, since this enzyme would hydrolyze the 5,10-methenyltetrahydrofolic acid formed. 'See J. Rabinowitz (Vol. VI) for the purification of formiminotetrahydrofolic acid cyclodeaminase from Clos~rldia extracts.

790

ENZYMES OF PROTEIN METABOLISM

[105]

Purification Procedure Steps 1 to 4 are the same as presented above for the preparation of formiminoglutamic acid formimino-transferase. To obtain a preparation of the cyelodeaminase relatively free of the formimino-transferase (see the table), the enzyme mixture is incubated at p H 10.5 in an a m m o n i u m hydroxide buffer. Under these conditions there is a relatively rapid inactivation of the formimino-transferase; some inactivation of the cyclodeaminase also occurs, but at a considerably slower rate. The supernatant solution obtained in step 4 is mixed with 0.05 vol. of a 1 M a m m o n i u m acetate-ammonium hydroxide buffer (pH : 10.5, measured at 2°). After 2.5 days the solution is transferred to a beaker which is placed in a vacuum desiccator over dilute sulfuric acid for several days to remove most of the ammonia (preparation 5'). The entire procedure is carried out at 0 ° to 2 °. Preparation 5' can be stored at --20 ° for I month with a loss of 10 to 30% of its activity. Properties Formiminotetrahydrofolie acid cyclodeaminase is markedly stimulated by K ÷ or NH4 +. The K~ for K ÷ is approximately 1.3 X 10-3 M ; for NH4 ÷ the K~ is approximately 1.8 X 10-8 M. The Km for formiminotetrahydrofolic acid is 17.5 X 10-5 M in the absence of added K ÷, and 2.6 X 10-5 M in the presence of K +. The activity of the enzyme increases with increasing p H ; a definitive p H optimum has not' been determined, since the enzyme assay is unsuitable at a higher pH, owing to the alkali lability of the product.

[ 106] NS-Formyltetrahydrofolic G l u t a m i c Acid Transformylase f r o m

Acid H o g Liver I

NS-Formyltetrahydrofolic acid -{- L-glutamic acid ~ Tetrahydrofolie acid + N-formyl-I,-glutamic acid By MILTON SILVERMAN Assay M e t h o d

Principle. The method is based on the measurement of the tetrahydrofolic acid formed. The assay employed for tetrahydrofolic acid 1 depends on quantitative conversion of this compound into p-amino1M. Silverman, J. C. Keresztesy, G. J. Koval, and R. C. Gardiner, J. Biol. Chem. 226, 83 (1957). Presentation of the above material is based on this paper.