Physical and Kinetic Properties of Dihydroorotate Dehydrogenase from Lactobacillus Bulgaricus

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Dissertations and Theses Dissertations and Theses

8-1-1969 Physical and kinetic properties of dihydroorotate dehydrogenase from Lactobacillus bulgaricus

Craig David Taylor Portland State University

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Recommended Citation Taylor, Craig David, "Physical and kinetic properties of dihydroorotate dehydrogenase from Lactobacillus bulgaricus" (1969). Dissertations and Theses. Paper 62. https://doi.org/10.15760/etd.62

This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. AN ABSTRACT OF TRE THESIS OF Craig David Taylor for the Master of

Science in Biology presented August 29 , 1969.

Title: Physical and Kinetic Properties of Dihydroorotate Dehydrogenase

from Lactobacillus bulgaricus •

APPROVED BY MEMBERS OF THE THESIS CO Mt-江 TEE:

David McClure

Dihydroorotate (DRO) dehydrogenase catalyzes the oxidation of DHO to orotate in the pyrimidine biosynthetic pathway. This enzyme was originally isolated from a bacterium, Zymobacterium oroticum, which would ferment orotate as a Bole source of energy. This adaptive catabolic enzyme , which catalyzes the reductiqn of orotate to DRO in an efficient pyridine nucleotide-linked reaction, has been extensively studied by several workers. Until recently, no study has been carried out on the enzyme which catalyzes the reaction in the biosynthetic direction. Pre- liminary studies have shown that the biosynthetic enzyme in Esherichia 쭈추추 and a pseudomonad is not capable of reducing orotate.to DRO by a pyridine nucleotide-linked reaction. These results suggested that there may be significant differences between the catabolic and biosynthetic enzymes.

In the present study biosynthetic DRO dehydrogenase from 꼴담0 bacillus bulgaricus was investigated on the basis of physical and kinetic properties in order to compare the enzyme with the extensively studied catabolic enzyme. The stoichiometry exhibited by the DRO oxidase activity of the biosynthetic enzyme and the absorption spectrum suggest that biosynthetic DHO dehydrogenase is a flavoprotein. Thin layer chromato graphy of the flavins extracted from the enzyme and reactivation of apo enzymes specific for flavin mononucleotide or flavin adenine dinucleotide have shown that the enzyme contains flavin mononucleotide.

The demonstration of enzyme-catalyzed sulfite autoxidation suggested that iron is present and is involved in electron transport. Inhibitor studies have shown that the enzyme contains sulfhydryl groups and the inactivation of such groups halts internal electron transport early in the sequence.

Kinetic studies were carried out including the·determination of the

Km for dihydroorotate t Ki for orotate t and the pH optimum. The kinetic behavior of the enzyme in the presence of various inhibitors suggest

that the essential sulfhydryl groups reside at or near the active site.

Ammonium sulfate was found to enhance the activity of the enzyme.

Evidence presented suggested that this phenomenon is probably an un specific anion effect in which the rate constant for the breakdown of

the enzyme substrate complex is directly affected. A possible scheme of the internal electron transport of biosyn thetic DilO dehydrogenase was presented. using the data from this thesis and additional evidence from studies carried out by other workers on similar enzymes. A summary of the physical and kinetic properties of biosynthetic and catabolic DRO dehydrogenase was presented and a detailed comparison between the two enzymes made. PHYSICAL AND KINETIC FROPERTIES OF DIHYDROOROTATE

DEHYDROGENASE FROM ’ LACTOBACILLUSBULGARICUS

/ by

Craig David Taylor

A thesis submitted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE in BIOLOGY

Portland State University 1969

짧랬폈 It웠많뼈 $?Jif ￡ TO THE OFFICE OF GRADUATE STUDIES:

The members of the Committee approve the thesis of

Craig David Taylor presented August 29 , 1969.

Herman Tavlor. Ch윌릎균

David McClure

APPROVED:

Earl F'isher Jr. Head Department of Biology

L. Roberts Acting Dean of Graduate Studies

August29. 1969 TABLE O:F C(퍼:~T’ El~T:3

PA 김 E

T과BLES LIS ,T OF .. • to ...... :vi

LI 전’r ιri. OF’ ?IGURES to . . c ••• ...... i.

IN’TRODUCTION .. e • .. “ • .. •• .. • .. •• a .. ••• l

MATERIALS AND 앤 ET Il ODS ‘ .. . ..fj...O...... c..~ 5 0 호 gHrl1 Sm.. • ‘•• . .. ‘...... 던 . . … i ‘ Orεani.딩m. Grmvth and Harvest of the .. , . . . ‘ ...... ‘~ II .. 5

Preparation of Cell Free Extracts .. • • • 0 • I) ...... t 7

Purification of Enzymes ...... ~ ...... c ~ • 7

Dehydrogεnas 당 ~ 톨 DBO ...... •••• .. .. • ’ ‘’ •‘ ι 1

NADPH Cytochrome c Reductase. ‘’~ ..... e ..... fJ 9

p D‘ -amino Acid Oxidase. .. .. • ...... ” ‘ ’. • n Enzyme Assay 딩 and Chemical Detξnninations J .. .. . •• " 9 ‘ i

~ ’깃 F' erricyanide Assay. " 0 • • • • ...... eo • .. " .. ’‘ .'

z‘ 6-Diζhlorophenolindophenol and CytochrolTI깐 C Rξ'duction Assays III " • ,$ ... .. • .. .. • ., • ., … g ‘ 10 r.

A강 rαbi.c 1.강 Assay .. .. • • .. ‘ 9 ...... " .. . ..

Convε~rtln 설 Optical Density eban월 e int。 Micromolar Changes ‘ • ~ • .. .. •• .. .. • • " 10

仁 or StuJte 잔 ρ ι 에 Iuhib ::i,‘ ‘ • .. • ...... " 10

Aut 낀 x :i. dation. 톨 Sulfite ,. co ., ...... ,(0 • ...... 10,

1、UDPH R 걷 ducta.s e Assay of Cytochrome c .. 。 ’“ a ‘ .. 11

Assay of D-“ ε‘1’nino Acid Oxidas깐...... 톨 ., • 1.1 i.·V

.PAGE

Measurement of 센l.e Rate of 0겼Y8 상 n Consumption, Orotate Formation, and Hydrogen Peroxid쉰

Productionu ~ ••••• • .. .. • " ~ • ~ 0 II • .. • 11

Chromatαgraphy" Flavin Analysis Using Thin Layer • . . o 13 Flavin Analysis Using apo-NADPH Cytochrome c apo-D-A니mino Reductase and Acid Oxidase G .. " " • 14

Commercial. Sources of Chemical핀 and En강 ym딪 So • 잉 II -". t ll~ f " 1/

RESULTS" •• ‘." .. . • " " " II •• " • .. •• e e ~ c ~ .. o 16

Dehydrogen 징:.s e F.’ lavoprotE쓰:t n~ Evidence for mIO Being g .“ •" 16

Aerobic Pr‘잉 duction of Hydrogen Peroxide, ~ . ~ II • ‘·. 16

Sp 앉 ctrum Dihydr인 orotate Dehydro짚enasc " Visible of e " " .,17

Qualitative Analysis for FIaγin. · " . B .. ’ . " "• \'I " ]‘7

Effect of pH •••• .. • e " t • " " " ~. .. .. 22 . . . . . • ‘ · O r.- ξitate 갚앙 Determinati.on of Km for DRO and Ki for of ~. .. '" l • (l

Effect of Ammonium Sulfate .. •• • • • 0 " co •• * II "• • • 32

Inhibiε。 rs" Effect of Sulfhydryl • 톨 ‘ It 6 ~ 36 " . " '" e II * ’ ”

Indirect Evidence that Iron is Invclv장~d in 턴딩 t 앉 lyn :i.설 e ~ 1~8 @ ‘’ Evidence Relating to the Flow of Electrons in

Biosynthetic DRO Dehydrogenase e .. •• ‘ e .~ t1 II .• • 0 • 50

R앙 duction of Various Electron Ace단ptors by Dihydr낀 orota. te Dehydro짝ellase III •• e • : .. .. . ε • 55

DISCU 전 SION ~ /I • e f!“D •••• II • • • • ‘~ • •• , " .. II .. " of 57

Transport~ 。 Internal Electron .. t' .. •; II " e .. “ II .. ••• .. 57

D 단 hydr 。앉깐 na 암암"' Catabollc DHO j. • 'I .. C • 훌 .. ¢ • • 57

Xanthine Oxida잔 e e ~ .. .. • • 훌 양 .. c .. 0. • ‘. " ~ if • ’ .. 58‘’

Biosynthξtic D 딩 hy 값 rι} 장원 nε:'8 굉 ι DHO .. .IJ .. 5 당 . “ II II ‘• C Olπpar 초 son of Catabolic and Bi。잔ynthetic·DHO Dehydrog앙，n a. sε. ‘‘ 61 v

F‘AGE

SUM1'1ARY 4O •• ‘ 1I ~ “• ’~ -. .. ., .. • .. ., . • • ‘• ., • ., • I;l . . " .. 64

REFERENCES • 。. ~ II • " .. II • .. It fJ e •‘ @ eI ., “” ’ , • ‘ .. 65

APPENDIX • • .. ~ (I (l II • • ., 0 • ~ • .. s ~ • $ • ...... • • • ‘ 68 LIST OF TABLES

TABLE PAGE

1 GrαNth Hedium for 후짧앓앓확싫혹싫꿇짧싫짧 •• ., • •• • 6 6

II Thin Layer Chromatography of the·F’lavins Eχtracted

from Biosynthetic Dihydroorotate Dehydro앉enase Q • II . 23

III Effect of the Flavins from Biosynthetic Dihydroorotate

Dehydrogenase on Reactivation of a항。-NADPE‘.、 Cyt c.-

chrome c Reductase 강nd apo-Afuino Acid Oxidase. • II. 2 각

IV Effect of Salts on Biosynthetic Dihydroorotate Dehydr。’‘

~ genase Activity. … 6 ••••••• (> •• 5 .. • .3 5

V Reversal of Sulfhydryl Inhibition by Mere강ptoethanol • •• 49

VI Effect of Tiran and p-Hydroxym당 rcuribenzoate on Dihydro

orotate Dehydrogenase Activity with Various Electron

Acceptors ••••••• ‘ .... II tI • ., •• It ., •• c 54

VII Rate of Oxidation of Dihydroorotate to Orotate by Bio

synthetic Dihydroorotate Dehydrogenase using

Various Electron Acceptor~ ‘.. tl •• c •• , 0 •• .56

VIII Properties of Catabolic and Biosynthεtic Dihydroorotate

De}i ydrogena딩 e. • • •• 0 0 ’ • II ••• ~ e • • • ‘. 62 LIST OF FIGURES

FIGURE PAGE

biosyτlthetic 1 Pyrimidine pathway in microorganisms.. .. ¢ • . . 2

2 ‘ The stoi강hiometry expectεd if·dihydrooroεate dehydro....

genase i.s a flavoprotein .. r...... • e .. •• .. l6

3 Comparison of th당 oxygen uptake 당nd hydrogen p(~rαxide

producti.on duri.ng conver딩 ion of dihydro‘)rotate

to or6tate by bio딩 y n. thetic di‘ hy닙I.roar. at 값 te dehydro""

gena.se •• II ‘ eo. •• .. .. II .. • e • ‘ •• 8 ‘ .. .. 0 18

4 Absorption spectrum αf biosynthetic dihydroorotate

dehydrogenaseo .. .. ~ .. • ...... ‘ ...... 20

5 The effect of pH on biosynthetic dihydroorotate dehydro-

gena5e activity. • e • •• ...... 0 25

6 1' hε effect of arotatε concentration on biosynth윈 tic

di.hydr ooro tat잔 dehydrogenase activity& • to .. 2 • t "' 27

7 The eff당 ct of orotate concentration on Krn for dihydro-

orotate. ’ ., .. ••• .. ’. e .. ••• .. .. • • • ••• , 30

8 The effect of ammon칩lID sulfate on biosynthetic dihydro-

dehydro딩 enase orotate activity ••• e • … . . . . 33 ‘

9 The effect of ammonium sulfate on the pH optimum 강 f

biosynthetic dihydroor깐 taεe.dehydrogenasee • • ‘ .•• 37

10 Effec仁 of ammonium sulfate on th단 Km for dihydroorotate. • • 39 viii

FIGURE PAGE

11 The effect of mercuri c. chloride on biosynthetic dihy"",

droorotate dehydrogena딩 e clctlvity and protection

by dihydroorotate. ‘. e . ... . " . " . " " . •• e Ifl 12 The effect of p-hydroxymεrcuribenzoateon biosynthetic

dihydroorotace dehydrogenase activity and protec-

tion by dihydroorotate "••• “ ••"• • e “ ‘. • 43

13 Protection cf biosyntheti. c dihydroorotate dehydrogenase

from n녔~rcuric chlor. idε~ inhibiti잔11 by δrotate “ 껴 6 “· 14 lTd. tiation of 원 ulfite autoxidation by biosynthetic dihy-

dehydrogenas안 droorotate o • e "• • • " tl • • • II e 51

15 Internal electron transport in cataboli.c dihydroorηt 강 t 건

dehydrogena 앓 e" " " 0 "" " 8 .. tl • . " . . " .. .. e 57

16 Internal elect강 on transport in xanthine oxi.dase ., .. ~ e a " II 58

17 Proposed 딩 e때 uence of internal electron transpε.lrt in

biosynthetic dihydroorotic d 당hydrogenase ι •• " 59 INTRODUCTION

Biosynthetic dihydroorotate dehydrogenase (OHO dehydrogenase) cat훌lyzes th률 이tid훌 t10n of dihydroorot훌 t흩 (DfLO) tα 。 rotaξ 윌 in the pyri midine biosynthetic pathway. In studies carried out by Lieberman and Kornberg for the establishment of the pathway of orotate breakdown, catabolic DHO dehydrogenase was first isolated from Zvmobacterium ￡도암

닫드쁨. an organism capable of fermenting orotic acid. From conclusions attained in this and subsequent studies, by these and other workers, (1,2,3,4) the sequence of pyrimidine biosynthesis occurs as shown in

E‘igure 1.

Beginning with the work of Lieberman and Kornberg (5) a series of detailed studies were carried out on the induced catabolic DHO dehydrogenase. It was found that the enzyme catalyzes the reduction of oro- , ’ tate by NADH (5) , the oxidation of NADH by 。핸gen as well a8 the oxida tion of DHO by NAD'+ and oxygen (6).

Analysis on purified extracts (6) and crystalline enzyme (7,8,9) revealed that the enzyme was a non-heme iron. labile sulfhydryl contain ing flavoprotein. unique in the fact that the prosthetic groups, flavin adenine dinucleo다de (FAD) and flavin mononucleotide (FMN) were present in a molar ratio of one. Detailed equilibrium (10) , kinetic (6,7.8,9) electron paramagnetic resonance spectroscopy (8.11,12.13,14,15 ,16) studies. as well as investigations of subunit structure (17) have been carried out on catabolic DHO dehydrogenase. Throughout the period of study it has generally been presumed, 2 COO- ureidosuccinate carbamate HnN-C‘ COO Pi A r\ r\- 4l j. 뻐뺀2랙 NH"CO ,", A'T'P ‘ 6 r HI"‘ N 'OPO"H" :0댄 fziR 靈 tε3 c 획 ba 빼 l-P “ f촬sPz魔z뚫隱z뚫zz鐵z뚫靈;뚫z뚫뚫鍵a뚫廳魔z뚫짧z말amy펀~ 0δ%ggg/“CCmκC쇄했CCm쩍O댄i

[dihYdroorotas라 ~I{2

。 rotidine-5’ -P orotate

c 출 coi 2 했H+ o;

다써」 ”생 .때 펙터一 CO 따 • 2 원 ，야 뼈‘ ’넓

n 0 ATP + l\Tfl꺼

uridine-5’-p uridine-5’-triphosphate ~y.tJ._dine-~’ triphosphatg

Figure 추. Pyrimidine biosynthetic pathway in microorganisms 3 with acceptance (4 , p.126) , that catabolic and biosynthetic DHO dehydrogenase are the same protein molecule. Workers have recognized that catabolic DHO dehydrogenase is an adaptive enzyme (6,18) and have noted that the oxidation of DHO may not be pyridine nucleotide linked (6).

It has recently been stated that the more efficient reduction of orotate by NADH is not of physiological consequence in light of the biosynthetic reaction (16). However no attempt was made to isolate biosynthetic DHO dehydrogenase for comparative purposes. Utilizing the findings of Yates and Pardee (19 ,20) that the pyrimidine pathway in 혹· .5.맡초. was repressible, the laboratory of Taylor and Taylor partially purified biosynthetic DHO dehydrogenase from derepressed pyrimidine mutants of 혹· .5.와후 (21) .' They found that the particle bound 뀔. .£와1 biosynthetic enzyme differed from the 즘. oroticum enzyme in that it was not\ linked to NADH neither as an electron donor for the reduction of orotate nor as a NADH oxidase. Further data supporting this finding were obtained in the same laboratory from a soil pseudomonad (22). This organism, which was capable of utilizing orotate as a sole or supplemental energy and carbon source, produced a constitutive, particle bound, synthetic DHO dehydrogenase as well as synthesizing a soluble catabolic enzyme in response to the introduction of orotate.

After separation of the two activities by centrifugation, studies revealed that the induced, catabolic enzyme demonstrated activities simi. lar to those of the 죠· oroticum enzyme , while the biosynthetic enzyme had catalytic properties similar to that isolated from.람· 뜸곽. This general observation was later supported by other work (23,24). A soluble biosynthetic enzyme was found to be present in the anaerobic 4 organism Lactobacillus bulgaricus. This organism requires a pyrimidine precursor for growth (25) and was found to produce comparatively large amounts of biosynthetic DHO dehydrogenase upon derepression by starva- tion for. pyrimidine (26)'. The DHO dehydrogenase has been extensively purified from extracts of derepressed cells in this laboratory.

The object of this study was to determine the general physical and kinetic properties of biosynthetic DHO dehydrogenase.from1. 뱉후· garicus so that a comparison could be made with the catabolic enzyme isolated from Z. oroticum- 5

MATERIALS ’ AND ‘ METHODS

Or~anism

Lactobacillus bul~arieus 09. ATCC 13866. This organism requires a pyrimidine for growth. Ureidosuccinate, dihydroorotate, orotate, or uracil satisfy this requirement. 6

TABLE I GROWTH MEDIUM FOR LACTOBACILLUS BULGARICUS

l'Iaterial Amount glliter

Vita'min-free casam1no acids 5.0 Casitone 10.0 K_HPO. 0.5

KH_PO:6 “‘2 ~4 0.5 Glucose 20.0 Sodium acetate ·3H~O 10.0 ~g~?4·7H20 - 0.2 NaCl 0.01 바1S0 ， 4.~• 4HU. 2... 0 0.01 Trisodium citrate 0.25_

mg/liter ζJ------,nu Adenine C 」 nu Guanine 껴 nu Xanthine J AU q cJ Pyridoxal ·HCl ‘ ’ nu Thiamine 4 ’ nu Calcium pantothenate 4 14 nv Riboflavin nu Niacin 14 nU tJ p-Aminobenzoic acid 끼 nU Pyridoxine 4 nu ,‘IJ Folic acid

14Jli ”다 r” ge’l •‘

Biotin 5.0 Vitamin B-12 2.0 Tween-80 leO ml 7 Preparation 앞 S를관 ￡똥투 Extracts (26) 1.. bulRaricus extract was prepared by resuspending the cells (0.3 g/ml) in 0.05 M Tris-Hel buffer (pH 7.6) containing 1 mM magnesium chloride and passing through a French Pressure Cell (American Instrument Co.) at 7,000 psi. The extract was supplemented with sodium orotate to a final concentration of 0.6 mM and centrifuged at 150,000 X g for 90 minutes at 00 in a Beckman model L refrigerated ultracentrifuge (Ti-50 head). The pellets were discarded and the clear yellow supernatant was used as the starting material for the purification of DHO dehydrogenase.

Purification 않， Enzymes (26)

탤으 Dehydrogenase. All of the following steps were carried out at

1. 0.025 volume of 1 Mmanganese chloridewas added dropwise t。 the 150,000 X g supernatant, with stirring. The stirring was allowed to continue fo호 15 minutes after the last portion of the manganese chloride was added. The extract was frozen at this point until further purification was undertaken.

2. The manganese treated extract was thawed and a sufficient amount of 0.2 M EDTA (pH 7.0) was added to give a final concentration of

0.01 M. The pH of the extract was then lowered to 4.1 by the addition of.

0.5 M sodium acetate buffer (pH 3.8) with stirring. The precipitated nucleic acid was removed by centrifugation at 10,000 X g for 10 minutes and the clear yellow extract passed through a Sephadex G-25 column equilibrated with AOV buffer [0.01 M sodium acetate buffer (pH 5.65) containing 0.6 mM sodium orotate. and 1 mM EDTA]~ This process was used 8

to exchange buffer systems and to remove the free f1avins a다d low mo1ec- ular weight compounds contained in the extract.

3. The enzymatically active fractions from the G-25 column were combined and solid sodium chloride was added to give a final concentra

tion of 0.15 M. Utilizing a batch procedure the enzyme was adsorbed t 。

Sephadex DEAE cellulose equilibrated with AOV containing 0.15 M sodium

chloride. The DEAE was washed once each with AOV containing 0.15 M

sodium chloride and AOV 'containing 0.20 M sodium chloride (wash'volume was about one thi~d-the combined volume of the G-25 extract). The

enzym~ was then eluted with a minimum amount of AOV containing 0.40 M

80diu뼈 훌hlo 합 de.

4. The enzyme fractions from the DEAE cellulose were pooled and

an equal volume of saturated ammonium sulfate (at 00) was 、 added with

stirring. Then solid ammonium sulfate was quickly added until the ex-

tract reached 80% saturation. Protein began to precipitate at 60% satu-

ration. As soon as the solid ammonium sulfate dissolved the precipitate

containing the enzyme was recovered by centrifugation. The pellets were well drained, and, redissolved in a minimum amount of AOV. 5. The enzyme fraction was clarified by centrifugation and quick-

ly applied to a 1.5 X 84 em Sephadex G-200 column equilibrated with AOV.

Fractions were collected at a rate of 5 m1/hour.

6. The most active Sephadex G-200 fractions we~e pooled and fur-

ther purified by polyacrylamide preparative disc electrophoresis with a

Polyprep apparat\훌 (Buchler Instruments, Fort Lee , N.J.) cooled by cir culating fluid from an external bath adjusted to -1°. The general pro cedure, preparation of the gels and Tris buffer systems were carτied out 9 as stated by Jovin 뜩 웰. (28). For enzyme stability 0.6 mM sodium orotate was included in all reagents and buffers. The samples were run through 2.0 cm of a 7% resolving gel at an applied current of 40 mae

The sample was eluted at a rate of 24 m1/hr and collected in a refrigerated fraction collector. For enzyme stability the fractions were adjusted to pH 5.6 with 0.5 M sodium acetate buffer (pH 3.8). The most active fractions constitute a 230 fold purification over derepressed cells (23,000 fold over ’ repressed cells) with a specific activity of about 6000 units/mg of protein (protein concentration was difficult to determine accurately at this concentration). Unless otherwise stated all studies were carried out on the enzyme at this state of purity.

뱉뿔갚 Cytochrome ￡ Reductase~ . NADPH cytochrome c reductase from brewer’s yeas~ was purified through the ethanol precipitation step and resolved for F뼈 by the method of Hass 똥 끝. (29).

딛「멜곽g 후닫혹 Oxidase. D-amino acid oxidase from an acetone powder

。 f pig kidney (30) was purified and resolved for FAD by the method of

Huennekens and Felton (31).

Enzyme Assays and Chemical Determinations

Ferricyanide 후뿔훨. DHO dehydrogenase activity was measured by a modified form of the ferricyanide reduction assay of Taylor and Taylor

(21). The assay mixture (3 m1) contained 300 μmoles of Tris~HCl buffer

(pH 7.6 to 7.8) , 36 μmoles of sodium dihydroorotate, 2.0 pmoles of potassium ferricyanide, and 1.0 to 6.0 units of enzyme. The reaction was initiated by adding DRO and potassium ferricyanide (0.5 ml total) to the

。ther components. The reduction of ferricyanid~ was followed spectrophotometrically with a Coleman Hftachi 124 double beam spectrophotometer ’

\ 11

techniques (32). Oxygen uptake was followed in a reaction mixture (2

m1) consisting of 200 μmoles of potassium phosphate buffer (pH 7.6) , 2

μmoles EDTA, 24 pmoles sodium dihydroorotate, 100 pmoles sodium sulfite,

and about 6 units of DHO dehydrogenase. Mannitol (300 pmo1es) or Tiron

(0.5 or 1.0 pmoles) were also added to some flasks. The reaction was

initiated by tipping in enzyme and sodium sulfite from separate side

arms.

웰뿔L 으￡ 뱉뿔뜩 Cytochrome ￡ Reductase. NADPH cytochrome c reduc-

tase w‘as assayed spectrophotometrically as described by Huennekens and

Felton (31).

쩔를흩요 요훌 D-amino 쩔뿔 Oxida홉 e. D-amino acid oxidase was assayed

manometrically in a Gilson differential respirometer by the procedure of

Huennekens and Felton (31).

Measurement 약 양ξ 혈뜩 앞 OxYgen Consumption, Orotate Formation,.

훨으 Hydro~en Peroxide Production. The reaction mixture (4ml) contained

400 μmoles of Tris-HC1 buffer (pH 7.6) , 48 pmoles of sodium dihydrooro

tate, 300 units of DHO dehY4rogenase (0.13 m1 of a 2390 unit/ml ofso1u- tion, specific activity 5 ,270 units/mg of protein) , and 20 μg.of catalase (when indicated). All components except DRO and catalase, which were added to separate sidearms, were dispensed into the main well of a

respirometer flask. The reaction was initiated by tipping in the DRO

and allowed to proceed with shaking at 23°.

Oxygen consumption was measured manometrically with a Gilson dif-

ferential respirometer (32).

The production of hydrogen peroxide was measured by a modification \

of the chromogen reaction commonly used in blood glucose analysis 12 (33,34.35) involving a perqxidase catalized oxidation of o-dianisidine dihydrochloride (3,3-dimethoxybenzidine dihydrochloride) by hydrogen peroxide. The chromogen mixture (50 ml) contained 7.89 pmoles (2.5 mg)

。 f o-dianisidine dihydrochloride, 40 ug of peroxidase, and 6 mmoles of sodium phosphate buffer (pH 7.0). The o-dianisidine solution was stored as a5 mg/ml aqueous stock and the peroxidase as a stock contain ing 1 mg in 25 ml of 0.12 M sodium phosphate buffer (pH 7.0). These stocks were stable at 40 for a few weeks. At the desired time intervals a sample (0.2 ml) was withdrawn from the respirometer flask specified for the hydrogen peroxide determinationand diluted into 5.8 m1 of the chromogen mixture. The mixture was allowed to react for 10 minutes and the intensity of the color measured spectrophotometrically at 455 nm in a Bausch and Lomb 505 spectrophotometer. The blank was prepared by add ing 0.20 ml of an'enzyme reaction mixture without DHO in place of the sample. A commercial solution of 30% hydrogen peroxide was standardized by the manometric measurement of the oxygen evolved upon the addition of catalase. Dilutions of freshly standardized solutions were used in the preparation of a standard curve for the o-dianisidine assay. A ca1cu lated amount of DHO dehydrogenase was added to the chromogen mixture used for the standard curves to more closely approximate the conditions of the assay.

Orotate production was measured by withdrawing samples (0.2 ml) at various times from a designated respirometer flask, diluting into 2.8 ml of 5% trichloroacetic acid (TCA). and measuring the absorbance due to

。rotate at 284 nm (DHO does not absorb significantly at this wave length). The protein concentration was not great enough to exhibit any 13 precipitation. The blank was prepared by diluting 0.20 ml of an enzyme solution containing no DHO into 2.8 ml of 5% TCA. A standard curve was prepared wiξh known concentrations of orotate added to DHO-free, enzyme- containing reaction mixtures.

Flavin Analγsis 월월ξ 탤꽁 느짧랴 Chromatography. Extraction of flavins from the 、 enzyme (specific activity about 3 ,000 units/mg of pro-

ε ‘~in) was carried out at room temperature by a method similar to that of l~ondo ξ￡ 휴추. (36) , the flavins being protected from light to minimize photolytic reactions. The enzyme, pooled from Buchler electrophoresis runs, were heated in a boiling water bath for 2 minutes , and the dena- tured protein removed by centrifugation. The flavins were separated from the solution by quickly extracting with 0.4 volume of water-satu rated phenol. l The phenol layer was separated from the extracted aque-

。us layer and the flavins returned to a small amount of fresh water

(0.1 to 0.2 ml) by shaking the mixture with two volumes of diethyl ether.

’rhe aqueous layer was subjected to a light vacuum to remove the residual ether. During the heating process some of the protein was broken down into ninhydrin detectable amino acids and peptides which were extracted into the phenol. \vhen in sufficient concentration, these contaminants caused streaking of the flavin spots during chromatography. A second rapid extraction with phenol usually separated the flavins away from enough of these compounds to yield sati닙 factory chromatographic results.2

lFor best results the phenol was surfaced with a 1/8 to 1/4 inch layer of water. Prior to use the εwo phases were shaken to an emulsion, allowed to separate, and the phenol removed with a Pasteur pipette.

2If a second phenol extraction yields unsatisfactory results it would best be replaced by eluting the contaminants away from the flavins lIt

The aqueous flavin solution \vas lyophilize.d t 。 ‘irynes8 and store갚 at

-20°. The flavins were dissolved in a min 초mum auiount of "‘rater and quali-

tatively identified by thin layer chromotography on cellulose gel HN

300 using the nucleotide solvent system t-강my1 alcohol, £εn‘ mic acid,

and water (3:2:1 v/v) or on Silica gel G using pyridine, 강 C 앙 tic acid

and water (10:1:40 v/v) (37)e

훤추뀔짧꼬 짧앓￡흥꿇 궐앓i릎앓 !]묘g댈싫윌꿇 ￡짧앓윌￡탔전폼:~ c 엎싫과￡꽁료짧‘ ￡짧많 찮짚O~ ‘ , 끓밸많꿇후훌 Oxid강닫르. After removal from the ξnzyme by the proce따~e

pr단viOtlsly ment.ioned, th 영 f1avins were qual갚.tati상당 1y analyzed from 꽉11-

quots of the boiled extracts (clarified by centrifugation if neξ

usin덩 th앙 apo-NADPH cytochrom당 c.reductase Csp 당 eific for. F’ r" n.~) and apo-

D-aminoacid oxidase (specific for FAD) assay systems (31).

Commerci. a조 Sources of Chemi c. a1s and 앓짧聽흘

앓짧월갇염과묘혹뭘싫월끊많앓n화 파g‘ Cellulose g앙 1 MN-300 , ~m-300 DEAE c 셉@

1u10 딩 e.

G. Frederick Smith Chemical Co. Tiron.

Mallinckrodt Chemical Co. Hannitol.

Hann 갚뚫認짧밀 L훨R프욕탤E파닫~. 2-mercaptoethanol, orotic acid.

Math단앓，Il...， 앓꿇짧끊 짧앞 앓끓. So파 urn 2~6‘ dich1orophenolindophenol.

황· 앓휠앓효， 합효흥많짧앓료요놈￡ 릎꿇짧많갚품후앓앓뀔많꿇효 .f..2. 0 Silica gε1 G.

힐잖 E짚효!낭 L뀔 ￡짧흘 ￡않많앓옳싫， 훌양，S. DEAE Sephadex A-50, Sephadex G-25 ,

G-200.

by thin layer i.덴 n exchange chromatography on HN DEAE cel1ulos 단 using

0 ,, 05 떠 hydrochlorie acid and then eluting the flavi.ns \·;iεh ()‘ 3. N hydrochloric 강 cid ‘ The hydrochloric acid is then quickly removed under vacuume 15

활웰혹 Chemical ~. Catalase (1 mg decomposes 3,000 pmoles of hydrogen peroxide/min); cytochrome c, Type III; o-dianisidine dihydrochloride; dihydroorotic acid; ethylenediamine tetraacetate. disodium salt, Sigma grade; flavin adenine dinucleotide, grade III; flavin mononucleotide, commercial grade; p-hydroxymercuribenzoate, sodium salt. 16

RESULTS

Evidence 혹으도 멜으 Dehydrogenase 욕발댈 혹 Flavoprotein

It was found early in the development of a purification scheme

for the biosynthetic enzyme from~. bulgaricus that the bright yellow color of 'the crude extract (150 ,000 X g supernatant) was due mainly to free flavins rather than flavoproteins. Passage of the yellow manga-

nese-treated extract through Sephadex G-25 resulted in relatively color

less active fractions. This observation left open the question of whether or not the biosynthetic enzyme was a flavoprotein.

Aerobic Production ￡혹 Hydrogen Peroxide. It is generally accepted

(9) that flavoproteins which catalyze oxidase type reactions reduce the

。핸gen to hydrogen peroxide. Since 1.. bulgaricus DHO dehydrogenase is capable of catalyzing the oxidation of DHO aerobically, the reaction

might be expected to~proceed as shown in Figur~ 2 if it is indeed a

flavoprotein.

패 o ι •// 핵。rotate

sL14핸 야 야 .파 r n reduced flavoprotein

catalase ~ H20~ o 1/2 O2 + H20 2 Figure 1. The stoichiometry expected if dihydroorotate dehydro genase is a flavoprotein.

An experiment was carried , out as described in Methods in which the rate 17 of oxygen consumption, orotate and hydrogen peroxide production were measured. If biosynthetic DHO dehydrogenase is a flavoprotein one would expect the stoichiometry of Figure 2 to be followed, namely the rates of oxygen consumption, orotate and hydrogen peroxide production will be equal. Also, if catalase were added at the beginning of the experiment, the hydrogen peroxide formed would be· immediately broken down into oxygen and water as shown in Figure 2. This occurrence would

decrease the observed oxygen consumption by one half. The results shown

in Figure 3 show that the stoichiometry exhibited by DHO dehydrogenase

is typical of a flavoprotein. The hydrogen peroxide was stable in the

reaction mixture as shown by the convergence of the curves resulting

from catalase added at the beginning and the end of the experimental

period.

Visible Spectrum ￡￡ Dihydroorotate Dehvdrogenase. Upon further

purification the characteristic yellow color of flavoproteins became

evident in concentrated fractions. Figure 4 shows the visible spectrum

of the most purified fraction of the enzyme from the preparative disc

electrophoresis separation. The spectrum is typical of a flavoprotein with distinct maxima at 380 ,466 nm and minima at 350,416 nm. There is a

noticeable shoulder at about 490 nm similar tothose present in other

flavoproteins (38,39 ,40 ,41 ,7,8,9,). The ultraviolet absorption peak observed for flavoproteins between 265 and 280 nm is obscured in this

preparation due to the presence of orotate which has a maximal ab~orp

tian at 282 nm.

Qualitative Analvsis 동양 Flavin. Qualitative analysis for flavins

(see Methods) carried out on pooled preparative disc electrophoresis 18

￡효옳앓룹 료. Comparison of 삼 Ie 0)ζygen uptake and hydrogen peroxide production during conversion of dihydroorotate t 。。 rotate by biosynthetic dihydroorotate dehydrogenase.

o r- 1"" ‘g

o It')

o ‘ t 짧川넓 ι 쩍싫뻗 m M @앓 뺏뽑흉없

。 t애

'------L.--.-_‘~‘_. .上 Q C깐 잉 C ‘애 o S 많 f 때짧딸짧 JI 짧 19

Figure 후. Comparison of the oxygen uptake and hydrogen peroxide production during conversion of dihydroorotate to orotate by bio synthetic dihydroorotate dehydrogenase.

The reaction mixtures and procedures were as stated in Methods. S펴yr뼈매 o따1s잉: (ωO이) ’ total μ매mo따lea of 이0 1핸연gen cons’i빼1 a랴t the arrow; (e> , total μmoles of oxygen consumed when catalase was present from the beginning of the experiment; ~口)， total }JIllo1es of o.rotate produced; (6) t" total μmoles of hydrogen peroxide produced.

/' 20

짧짧표융 i. Absorption spectrum of biosynthetic dihydroorotate de hydrogenase.

·많(} r-a-야g‘홉1 -'""'"월，......

c그 c::> L..r.>

/t￡gI홉‘엇‘

、‘￡n렇..~ c::> ιζ그 특r 룹c

c..，~ --‘혈2:밍 ζ3 μl...J 실~ ___J LL..I ξ=풍$:· 번S룰옆"""웰ii·i

C그 c。 rγ 〉

--람 ·& 여 #C [} 、。 c、‘ c 。 38 뼈냄했썼 aS8 뻗

*“

~'，' .~~ ...，~ ... t"~'~~'~"~":'.~""-"_.~、-，.-;--‘”’ ••_."," ..... -.-“ "":"---~ • e . -. ←、、’‘t“ η V~';'-' π ， -ι ‘ -ι ‘ p. ‘ ~，，'!.J，TT π ‘’ ~!'!'-'-"':'-"'r"~'~_r::"'::-γ...... ~"'l、、‘ γν π':-"'~~ .. ""~，‘”’‘’--’， •. ~ 2~ ‘ ” 21

Fi2ure 후. Absorption spectr빼 of biosynthetic dihydroorotate de hydrogenase.

The spectrum was obtained with the most active fraction of the preparative disk electrophoresis preparation. The sample con tained about 0.6 mg of protein/ml and had a specific activity of about 6,000 units/mg of protein. The solution also contained 0.6 mM sodium orotate. 22

fractions Bugge앉 t that biosynthεtic DRO dehydτ 。낌원 nas 던 contain 딩 FH센@

Results of the thin layer chromatographic analysis are shown in T~)l뒀

II. In both thin layer systems u딩 ed flavin obtained from boiled extract mj.grated as a single spot with un Rf very close to that of FNN standards.

The unknown"migrated as FMN when mixed 꾀Lth chromatographica호 ly purifie산.

FAD and as a sin 당 Ie spot when mixed with chromato당 raphically purified

EMN. Flavin "adenine dinucleotide was not noticeably hydrolyzed to FMN

했hen carried through the same heat treatment and extraction procedur‘ E

1양s the uuknm·lu.

Further supporting evid딩 nce is shown in Table III e Thε flavi.ns in

";1적~e unknown are capable of ‘ reactivating apo-NADPH‘ cytochromε ε reductase

~ut not apo-D-aminoacid oxidase, suggesting that on꾀r Ii멘싼~ is present :i.n the biosynthetic enzyme.

~.2f.2끓

The variation of enzyme activity with pHis shown i.n Figure 5. representing two experiments performed 10 days apart. The pH optimum most probably lies between 7.6 and 7.8, the determination of which is made somewhat difficult by the fact that there is no large change (about

17%) in the enzyme activity over th단 ξntire pH range. measured.

It is possible that pH curves such as those in Figur단 5 represent a composite effectof pH on Km (Hichaelis constant) as 'veIl as on enzyme activity. Suffic:i. ent increases in Km with pH can occur such that the substrate concentration us 당 d in th~ assay is no longer saturating, thus

causing a decrea 딛 e in reaction ra~쉰 (1~2) e Th :i.s LtD‘ litation can be ov딛 r-‘ come by obtaining enough data at each pH to obt8. in L:!.neHeav81'“’Burk plots 23

TABLE II THIN LAYER CHROMATOGRAPHY OF THE FLAVINS EXTRACTED FROM BIOSYNTHETIC DIHYDROOROTATE DEHYDROGENASE

t~amyl alcoliol~formic Pyridine - acetic 용훌10 륭 ”훌훌훌r. (St훌- acid 를 W훌t훌 r. (률 ta- tionary phase. 뼈 300 tionary phase, silica Sample cellulose gel gel G)

Rf Rf Riboflavin 0.675

Flo때 0.528 0.524

FAD 0.260 0.720

Unknown 0.526 0.540

The solvents and procedure were as stated in Methods. 24

TABLE III

EFFECT OF THE FLAVINS FROM BIOSYNTHETIC DIHYDROOROTATE DEHYDROGENASE ON REACTIVATION OF apo-NADPH-CYTOCHROME c REDUCTASE AND apo-D-AM1NOACID OXIDASE

Concentration of added Reaction Rate flavin in reaction mix ture; ml of unknown NADPH cytochrome c added. reductase D-amin6 , •, acid oxidase

OD __ .... /min μ10 'consumed/min PM 550’빼 2 FMN

0.0667 0.0365 --tII!'- 0.0533 0.0270 ..- 0.0333 0.0230

None 0.0165 FAD

0.333 8.3

None 3.7 Unknown

0.05 ml 0.0225 (equivalent to about 0.03 μM flavin)

0.10 ml 0.0280 (equivalent to about 0.06 따 flavin)

0.60 ml 3.6 (equivalent to about 0.36 μM flavin)

The preparation of the apoenzymes and the assay procedures were as stated in ~얼마ods. The unknown consisted of a he~t treated 1/10 dilution in water of a 3,000 때itlm1 solution of biosyn~hetic DRO dehydrogenase (specific activity about 6,000 뻐its/mg of protein). 25

과후§콰짧혹. The effect of pH on biosynthetic dihydroorotate dehydro‘ genase activity.

C-O c。

c、J cd

뺑* 넓R

-캉· ~

1li!

양타

l111

111

양 L_~_ .따

q (y) r、J .-• <::) 떼 η 때 박 η 꺼 쐐 「 때 에 H η 『『 베 떼 ” ‘ 샤 n H l n n I n a n , 케 ’ ” 염 U 펙 / μ 4 l 싸 애 뼈 얘 꾀 a 빼 4 、 4 R μ 베 M 닝 H / 씨 나 넙 U닙 U U l 뼈 ” 며, 생 N” U 낭 닝 l l 해, u ‘ U m * @ ‘ 26

Fi~ure 흑. The effect of pH on biosyntheticdthydroorotate dehy drogenase activity.

The reaction mixture contained 300 μmoles of Tris-maleate buffer (pH 6.8 to 7.3) or Tris-HCI buffer (pH 7.2 to 8.6) , 2 μmoles of potassium ferricyanide, 36 μmoles of sodium dihydroorotate, and 0.10 ml of a 1/100 dilution in AOV of a 3,800 unitlml solution of enzyme (specific activity about 6,000 units/mg of protein). The reaction was initiated by adding enzyme in 1.0 ml of AV to the remaining reagents. Ferricyanide reduction was followed colori metrically with a Klett-Summerson colorimeter .(#42 filter) for 12 minutes. The pH of the reaction mixtures were measured at the end of the reaction period. Symbols: 0그). Tris-maleate buffer; (톨;) t Tris-HCI buffer.

/ 27

￡않월끔흘 ，효. The effect of orotate concentration on biosyn… thetic dihydroorotate dehydro딛 enase activity.

T‘ 양〉

i '"

F’,- IEF E

F ‘•-‘、 L↓」

ζ흘 •• ::r:: C그 ....- 、‘·‘/

l 뺀/033noqHd 31 \1 1쁘? 」 O Sf1OW 랜뇨 ,_,.1__ 한I 28

Figure ξ. The effect of orotate concentration on biosynthetic dihydroorotate dehydrogenase activity.

The reaction mixture (3 ml) contained 300 μmoles of Tris-Hel buf fer (pH 7.7) , 2 μnoles of potassium ferricyanide, 1.2, 3.6, 12.0, or 48 μ，moles of sodium dihydroorotate, 0.09, 0.24, 0.39, 0.69, μmoles 1.29, or 2.49 of sodium orotate, and 0.15 ml of a 1/100 in r AOV of a 2960 unit/ml solution of enzyme (specific activity about 6,000 units/mg of protein). Several series of reactions were set up toobtain Lineweaver-Burk plots, each at a given orotate concentration, in which the DHO concentration varied from 0.4 to 16 mM. The reactions were initiated by adding enzyme , in 0.5 ml of AV , to the remaining reagents and followed colorametrica11y with a Klett-Summerson colorimeter‘ (#42 filter) for 12 minutes. Symbols: orotate concentration in the reaction mixture 률 (0) , 0.030 mM; φ~ ， 0.080 toM; (I口>. 0.13 mM; (e) , 0.23 mM; “~， 0·.4,3 mM; (_), 0.83 DIM. 29 from which the maximum velocity (Vmax) is calculated. Vmax is then plotted against pH. The pH optimum curves for DHO dehydrogenase pre pared in this manner were found to be identical to those prepared under the conditions of Figure 5.

Determination of Km for DHO and Kifor Orotate The Lineweaver-Burk plot shown in Figure 6 shows that orotate is, as might he expected, a competitive inhibitor in the forwardreaction

(the oxidation of DHO) of DHO dehyd~ogenase8 Because the apparent Km increases with increasing concentration of a competitive inhibitor, the determination of the actual Km is complicated by the presence of such compounds" ‘ This is the case with DHO dehydrogenase. Sodium orotate

(0.6 mM) is present by necessity both in the concentrated Buchler e1ec trophoresis fractions and in their dilutions which areused in the ex periments. It can be shown from relations derived from basic concepts

(43,44) (derivations in Appendix I) that the apparent Km increases 1ine ar1y with concentration of the competitive inhibitor. If this is the case then one should be able to plot the apparent Km vs concentration of

。 rotate 하ld obtain a straight line which can be extrapolated to the actu al Km at zero concentration of orotate. Figure 7 shows the results of such a relationship from which an extrapolated Km of 0.5 mM is obtained.

The Ki (inhibitor constant) for orotate was found to be about 0.1 mM by plotting 1/vi vs 1/(S) , (5)/Vi vs (5). calculating Ki from slope or intercept equations (44, p.151) and calculating for Ki from the data in Figure 5 utilizing relations derived in Appendix I. 30

￡옳효~.릎릎 2 The effect of orotate concentrati. on on Krn for dihydro- orotatε.

C융 -r-‘----,--'

.... """ 짧\ 。。

4’-、 ‘억. 용며 r-얘

‘g 뿜밟>< ]넓 l 웰뺑 lF ~ 뼈〕 앓

{]

i 하 n o l t') C‘i r- o 빠 ‘ 얘 I, n T I 川 l ---Fv l 뼈 I H --/넓 ! A 빼 ,ιn v ” 4 뼈표뻐랩뼈뼈냄

‘ 31

Figure l. The effect of orotate concentration on Km for dihydro orotate.

The experimental conditions were identical to those of Figure 6. 5y뼈ols: (e.이. represent results of two different experiments.

기 32 Effect of Ammonium Sulfate

In the dεvelopment of the purification scheme it was discovered

that the presence ofa1Thl1oniumsulfate markedly enhanced enzyme activi.ty

as measured by the ferricyanide reduction as 앉 ay (26). Figure 8 shOtvs

that this εffect increases with increasing concentrations of ammonium

sulfate, saturating at about 1.4 M, and decreasing from that point on.

As shO\\7n· in Table IV equimolar concentrations of other salts exj.bited

similar but less marked stimulation than does ammonium sulfate.

Similar εffects haY당 been observed by other workers (45 ,46 ,42 , P~

443). Massey found that the presence of various multivalent anions re-

suIted in a shift of t.he alkaline branch of the pH optimum curv딩 。 f

fumarase further towards the alkaline range. ’The affinity of the en-

zyme for its substrate remained unaltered (46). He predi.cted that the

anion binds to a basic group adjacent to the ionizable groups in the

active site that determine the pH curve~ Whether or not ~he group

affected in the active site is acid or basic, the suppression of the

adjacent positive charge will increase the pKa of th원 group~ thus shift-

ing the corresponding branch of the pH curve t O\i/ards the alkaline o

Another phenomenon '\las observed by Webb and MorrO\v (47) in which the

height of the pH curve Has increased in the presenee of the anion. 1’he

form and positions of the curves did not change and there 'vas no effect

of the a.nion on the affinity of the enzyme for it 딩 substrate. They 1n-

terpreted these results to mean that the anion directly eff~cted the

velocity constant of the breakdown of the εnzyme-substrate (ES) compI션X

to enzyme + product [ k2) , 1 33

따 셀 s e o Lu--i 압 후홍뀔짧올 효. The effect of am따m때11 퍼 n o t n a 야 f V/ thetic dihydroorotate dehydrogen~se 』 •

。 ....,------' N,

줄) 잉 .F 법휩」그이

홉그-응

어 .p art 릎댁 냐디 aσ- ∞ 」〈 .。 t 」 Z 벼익흐 Og # G•

ζ二-.증잭룹-‘-1.‘~~‘’--‘<_r~-1 %] 0 N ζ:> 0 a ￠폐 ""t - F

A.l. II\Ll8V 3 J\ ll \j 13~ 34

、「

Figure .용. The effect of ammonium sulfate on biosynthetic dihydro orotate dehydrogenase activity.

The assay mixture (3 ml) contained 300 μmoles of Tris-Hel buffer (pH 7.8). 2 μmoles of potassium ferricyanide, 36 μmoles of sodium dihydroorotate, 0.00, 0.15, 0.30, 0.60, 1.20, 3.20, 4.20, or 6.00 mmo1es of ammonium sulfate, and 0 8 10 m1 of a 1/5 dilution in AOV 。 f a 63 unit/m1 solution of enzyme (specific activity about 1030 units/mg of protein). The reaction ~as initiated with ferricya nide and followed colorimetrically with a Klett-Summerson colori meter (#42 filter) for 12 minutese 35

TABLE IV -

EFFECT OF SALTS ON BIOSYNTHETIC DIHYDROORQTATE DEHYDROGENASE ACTIVITY .

Salt added Relative ionic strength Relative activity

None 1.00

KCl ,1 1.10

NaCl l 1.14

MgC1 3 1.16 2 짜14 C1 l 1.27

Na2·""4... SO 3 1.33

MgS04 4 1.35 K2...... 4... SO 3 1.40

(NH .) ... 4'2/ ..,SO....4 3 1.70

The reaction mixture and procedure followed is described in Methods under the ferricyanide assay except , that 500 μmoles of the in dicated salt was also added. The enzYme consisted of 1.10 ml of a 37 unit/ml solution of combined Sephadex 뻐AE fractions (specific'activity 156 units/mg of protein). 36

Figures 9 and 10 show the results of experiments performed to see if the anunonium sulfate stimulation of biosynthetic ORO dehydrogenase activity follows either of the two mechanisms discussed above. As can be seen from Figure 9, the presence of ammonium sulfate does not appear to markedly shift the position of the pR optimum, but rather exhibits an increase in the activity of the enzYme throughout the range of pR values tested. Figure 10 shows that the Km for ORO is unaffected by the presence of ammonium sulfate. If the assumption that 뼈 툴 k_l/k is made l and holds true with ORO dehydrogenase it can be interpreted that ammon-

、 ium sulfate has no effect on the affinity of the enzYme for its substrate. It appears that the ammonium sulfate stimulation of DHO dehydrogenase would best be explained by the conclusions arrived at by Webb and

Morrow (47) , namely that the salt effects k2•

Effect of Sulfhydryl Inhibitors

Curve A of Figures 11 and 12 show the time course of inhibition of biosynthetic ORO dehydrogenase by the sulfhydryl inhibitors mercuric chloride and p-hydro핸~ercuribenzoate (p-RMB). These data suggest that if the sulfhydryl groups are not catalytically functional, they are at least located at or near the active site so that the mercaptide steari- cally or electrostatically inhibits the reaction. The rate of inhibition is quite slow, taking from 15 to 30 minutes to approach equilibrium inhibition. Since Ki is valid only at equilibrium conditions its determination for mercuric chloride and p-HMB would be tediousand probably

lThis inte~preta~ion was made under the assumption that of the relation E + S 뺨 ES 팍 E + P (where E 흩 enzyme , S• substrate, P• product and k • "rate constant) KIn • k _/k_. , -1·-1 37

￡짧끊￡L 혹. The effect of ammonium sulfate on the pH optimum of bio synthetic dihydroorotate dehydrogenase.

r ‘ -.

∞ m.

N ∞.

c。￥& r-: 빼R

낱 h•

다 .k

m L_~~_~~_ ..‘’__,.l 따. 낱 m 어 [ m 써때 n -콰 n 며내‘혀껴，짧 M ’껴 기껴배때，，야감싫μ m 까 빼 //- nNu%nu nUl4JRU 「 빠빼뼈 ”성 나닝 μ 해냥 앤냥 없 IINμ I U U 때 빼l 38

Figure i. The effect of ammonium sulfate on the pH optimum of biosynthetic dihydroorotate dehydrogenase.

The reaction‘ mixtures' and procedureswere identical to those in Figure 5 except that in the 'indicated curve, 500 pmoles of ammoni um sulfate was also present. Symbols: (0 ，口)，， 1ris-ma1eate buffer; (e,.), Tris-He1 buffer. 39

￡싫꿇융lQ.. Effect of ammonium sulfate on the Km for dihydroorotate.

σ3

Fl N 뿔뿔 F| 쩍벼 I ..,..... 뻗贊썼넓앓합빽넓혔빼쪽넓ι

--며 ~-----I o lη w 옆떤썩넓￥X

@、l

뼈 / μ 베 / 「 이 파 ‘ 1 l 「 l ‘ n 」 n n n 서” 서, ” n n n 1 n ” -- 나 N 띠 / l m사 4 4 l , ‘~M ” 」 ” • U \ ” d ·I ” u 니 」 ” 」 | U U 」 ” u u ” u U v ] v I l ‘l 40

Fi~ure 곽. Effect of ammonium sulfate on the Km for dihydrooro tate.

The reaction m!xt'ure (3 ml) contained 300 뻐noles of Tris-He! buf fer (pH 7.7) , 2 μmoles of potassium ferricyanide, 1.20, 1.68, 3.60, 12.0, 48.0 μmoles of sodium dihydroorotate, 500 μmoles of ammonium sulfate (where indicated) , and 0.10 ml of a 1/50 dilution in AO of a 2,500. unit/ml solution of enzyme (specific activity about 5,000 units/mg of protein). The reaction was initiated by adding DHO and ferricyanide and followed spectrophotometrically in a Coleman Hitachi 124 at 420 nm for 3 minutes. Initial rates were used. Symbols: (0) , ammonium sulfate present; (e) , ammonium sulfate absent. 41

찮&옳앓옳 후1. The effect of mercu~ic chloride on biosynthetic dihydroorotate dehγdrogenase activity and protection by dihydro orotate.

''----' 1--.‘，~， @

。 #

N ￡。·다띠一工효 ‘9 M

}{←-를 -..:.t N 요3 I갱::) 용므」·〈∞그〔)응‘

예3 냐。 버a 〔「}프특강L

∞

] L__---l- _ 정 L--L.. F.φ L‘」 L ll) i.t') 。F φ o -(닝 φ NIW/03JnOO~d 31 \f lO~O jQ S310Wrl 1~2

짧짧효윷 꿇. The effect of mercuric chloride onbiosynεhetic dihy‘ dro 。 rotate dghydrogenase aFtivity and pr。 tectiorl by dihydreor·ow tate.

The reaction mixtures (3 ml) contained 300 μmoles of Tris-HCl buf fer (pH 7.7) , 2 pmoles of potassium ferricyanide, 36 μmoles of sodium dihydroorotate, 0.1 nmo1es of mer‘ curie chloride, 0~20 rn1 of 1/50 dilution in AO of a 3 ,000 unit/ml solution of enzyme (specific acti:vity about 6 ,000 units/mg of protein). The mixtures also contained 2 μmoles of acetate and 0.012 pmoles of orotate from carryover of AO with the enzyme. In curve"A enzyme was added to the buffer and water follmved by mercuric ch10ridε (or water fo~ the con~rol， .curve C) at specified times (volume 2.5 ml). This mixture was allmved to incubate at room tempe.rat\lre for vari 。 us times and then the reac~ion initiated by adding DHO and potas‘ sium ferricyanide (0.5 mI total). In curve B (and its control) the DUO was present in the 2.5 ml incubation volume and the reac tion initiated by adding ferricyanide and water (0.5 m1 total). The reaction rates were followed spectrophotometrical1y in Coleman Hitachi 124 at 420 nrn for three minutes. 43

짧짧죠옳 후감. The εffect of p-hydroxymercuribenzoate on biosynthetic dihydroorotate dehydrogenase activity and protection by dihydroorotate.

--D.-.__~ 냈~월----꿇 릎E \ 내Q 그Q a 또[) 버 • nU』5 •fR ~}。」(] C 엽 。」 든 품

0.01

0.005 o 8 16 24 32 MINUTES OFINCUBATION WITH INHIBITOR 44

￡꿇꿇흩 .꿇. The effect of p-hydroxymecuribenzoate on biosynthetic dihydroorotate dehydr~될 enase activity and protection by dihydro orotate.

The experimental conditions were identical to those in Figure 11 except that 5 nm0les of p-Hl멈 were added in place of the mercuric chloride. 16 not accurate due to indeterminant secondary changes in·the enzyme over the long incubation period. Such eff닫cts are evidenced in curve A of

Figure 11 by the deviation from exponential decay of enzyme activity. A similar argument would stand for the difficulty of determining, by the method employed in Figure- 6 t whether these inhibitors are competitive or noncompetitive.

Curve B of Figures 11 and 12 shows that if the substrate DHO is present in the incubation mixture the rate of inhibition is very marked-

1y decreased. This data is kinetic evidence that mercuric chloride and

/ 야 떠 願 I F 랴 뼈 따 끄h , 뼈” 야 I 「 R , 4 t 단 핸 퍼” 짧 ” Q ·n --m --ι• s l 4 아 D P ,1 m. r ‘ m u 1 e e c e l 」 J L ‘, J 냉 L A r ‘ 、 ,. ‘ . ,• • ‘ that the sulfhydryl group resides within or very near the active site.

Since orotate is a competitive inhibitor, rapidly reaching equilibrium inhibition, it would seem likely that its presenc강 in 10짜 concentration tvould also protect the enzyme from sulfhydryl inhibition. Curve A of

Figure 13 shows this to be the case with mercuric chloride up to an orotate concentration in the incubation mixtur.e of about 0.45 mH. After this point 、 orotate begins to inhibit more than protect~ A similar but less marked effect is shmm in curve B after incubation with mercuric chloride for 16 minutes. Curve Cshmvs the degree of ;inhibition exhibited·by orotate itself in the absence of merc~ric chloride. Because of the small amount of orotate present in the enzyme solution for stability (which is therefore introduced into the reaction mixture) andthe results in Figure 13, the absolute inhibition in Figures 11 and 12 are slightly less (4 to 5% or less) than would be expected if orotate were not present. However this should not.alter any conclusions drawn from these data.

1••• 46

잎認，앞깊烏 후칭‘ Protection of biosynthetic dihydroorotate d e.hydrogen‘• 당 s c: f re..H [1 m 헌J: cur ‘ ie chlor:i‘ de inhibition by orotate.

r“ -.-…-‘_._-_. --‘-r‘ ",‘T

M 뿜옳썼 “ ‘뽑g빼‘‘ 넓.혐똘.뻗쩍넓혐넓쪽넓

r에 빼용넓h 뿔빼 쩍넓 [뻗불옐짧밟빼 넓‘ 품 씨빼 〔넓 뿔 쩍넓 ,..... 선밟

l l ,--1 ._ 띠.。 학.。 혀 잉 I펴 (,'1') F“‘ [) 。 영 o 。. .φ μ 씨 / -‘「 버 끼 때 η 맙 뼈 n n n n 「 얘 비 뻐 / l m 」 |‘ 서” -Jn 미 u 」 니 ” 」 U U U l t > ” 4 니 l 47

짧짧릎릎 꿇. Pr. otection. of biosynthetic dihy녕 roorotate dehydr‘。 gen ase from mercuric chloride inhibition by orotate,

Th~ reaction mixture (3 ml) consisted of 300 μmoles of Tris-HCl buffer (pH 7.7) , 2 pmoles of potassium ferricyanide, 36 pmoles of sodium dihydroorotate, 0.1 nmoles of mercuric chloride, 0.20 ml of a 1/50 dilution in AO of a 3,000 unit/ml DUO dehydrogenase solu- ‘ ‘ tion (specific activity about 6 ,000 units/mg of proteip). and 0.50, 1.0, 1.5, 2.0, 4.0, or 8.0 pmoles of added sodium orotate. 0.012 additional' pmoles·of orotate as well as 2 pmoles of acetate werecarried over.with the enzyme, In curves A and B enzyme was added to the buffer, \vat~r ， and a specified amount of orotate followed by mercuric chloride at a given time (volume 2.5 ml). This.mixture was allowed to incubate for 5 minutes (curve A) and 16 minutes (curve B before the reaction was initiated by adding DBO and potassium ferricyanide (0.5 ml total). The experimental conditions for curve C were the same as those for curve A except that water replaced mercuric chloride. The reaction rates were foIlα，'l ed spectrophot'ometrically in a Coleman Hatachi 124 atl 420 nrn for three minutes.

、 48

Further evidenc당 for the inhibition involving a sulfhydryl group is given in Table Vo After the enzyme is inhibited to the level ind.i- cated, incubation \vith 0.175 mN mercaptoethanol for 3 minutes reversed inhibition to within 90χ of full activity.

Indirect Evidence that I.ron is Involved in ea짧싫蠻

Fridovich and Handler (48) found that the non-감leme ion containing flavoprotein~ xanthine oxidase, catalyzes substrate dependent sulfite autoxidati단η via a frεe radical reaction~ They postulated that during norp'!al cat,alysis iron served an electron t. ransport function. resulting in a transient existence of the ferrous state (lf9) \vhich could inter당 ct +t+ directly with oxygen, forming oxygen fr원월 radicals: Fe + 02 --황

Fe++ ~- .0 - (49). Several lines of evidence were presented supporting 2 this postulate (50)$ Sincε that time non'’-heme iron containing and iron- free flavoproteins \"hich are capable of reducing oxygen to hydr。당en peroxide were tested for the ability to initiate sulfite autoxidation. How- ever, to date on~y three are known to catalize the reaction: xanthine

。 xidase ， aldehyde. oxidase (51)~ and catabolic DHO dehydrogenase (9)J all ofwhich are non-heme iron containingCflavoproteins. All three have been shown to form an enzyme bo~nd oxygen free radical by several criteria

(52). Further evidence has lead to the implication that the iron atoms in these three enzymes undergo idεutical valence changes and in fact may be bound to similarifnot identical ligands (9). If biosynthetic mID dehydrogenas 당 demonstrates similar oxyg당n free radical formation» this would provide good indirect evidence that i.ron is present and that th낀 이 mech견 nism of its action mi εht be similar or identical to that 49

TABLE V

REVERSAL OF SULFHYDRYL INHIBITION BY MERCAPTOETHANOL

-과 ”과 L & Treatment h 끄 O r ，러 찌 T·i ” , ”매 44 띠 ·『 패 없 1m m --• 따 Bnm f-or N。 ·i m, m L 에따 C)No inhibitor 따 빼 f b 꽤 1Q bvt M r cap μ← ‘ m않 않 야‘ 0 따‘ esen t e 댄 따 며

Wi4G wernu m:7rmT .... ~ ---띠l‘a‘~깅--“ pmoles DHO pmoles DHO pmoles DHO pmoles DUO 。 xidized/min oxidized/min 0χidized/min oxidized/min

HgC1 0.0991 0.147 2 pHMB 0.001+37 0.151

None 0.171a ,b 0.163~ 0.167U

--‘- The reaction mixture (3 ml) contained 300 μmoles of Tris-HCI buf fer (pH 7.7)t 2 pnio1es of potassium ferricyanide, 36 pmoles of sodium dihydroorotate, 5 nmoles of p-HMB or 0.1 nmoles of mercuric chloride, 0.20 ml of a 1/50 dilution in AO of a 3 ,000 unit/ml ·solution of enzyme (specific activity about 6 ,000 units/mg of protein) , and 0.438 pmoles of mercaptoethanol (where added). The inhibited and control rates (A and C) were obtained by a procedure identical to that used in curve A of Figures 11 and 12 except that the i.ncubation volume was 2.2 fil ana the incubation time with inhibitor was set at 5 minutes for p-H~m ， 15 minutes for mercuric chloride. The reaction rate of B was obtained by incubating enzyme, buffer and water with inhi.bitor (incnb-ation volume was 2.2 ml) for the specified time , adding rnercaptoethanol, and allow ing the mixture (2.5 ml) to incubate three minutes. The reaction "Tas then initiated by adding DHO and ferricyanide (0.5 m!). Rate D was obtained in the same manner except that water replae딘 d inhibitor. The enzyme was incubated in the buffer and water‘ mixture (total volume 2.2 ml) for a 15 minutes , b -5 minutes.

‘.. 50 of the three other extensively studied iron flavoproteins. Figure 14 shows results typical to substrate dependent , enzyme catalyzed sulfite autoxidation. The greatly amplified oxygen uptake over that in which sulfite is absent, and the quenching of the sulfite dependent oxygen uptake by the presence of a free radical ’'scavenger," mannitol, suggests that the biosynthetic DIlO dehydrogenas~-catalyzed reaction does indeed proceed by a free radical mechanism. Figure 14 shows that the ferric iron ligand Tiron (disodiu,m 1 ， 2-dihydr !J xybenz 당1. e‘ 3 ， 5-disulfonate) lnarkedly inhibits sulfite autoxidation, a phenomenon also ob~erved with catabolic DRO dehydrogenase (9) , providing further evidence that iron may be invσlved in oxygen free radical formation.

This laboratory has also demonstrated the presence of iron in bio- ‘ ‘ synthetic DRO dehydrogenase (26) by the use of the o-phenanthroline assay of Hass당y (53) in which the colored ferrous iron-o~phenanthroline chelate was measured spectrophotometrically.

뭘좌짧짧릎올앓꿇짧과옳 to 앓릎 꿇요꿇 요훌 짧앓찮짧릎 혹끊 짧꿇짧많짧짧￡짧않 앓앓앓0-

옳를감옆등옳

In active site studies, Fridovich and Handler with xanthine oxidase (/.9 J 51f) and Aleman and Handler with catabolic DRO' dehydrogenase (9) found that dye reduction and reduction of oxygen to hydrogen peroxide responded differently to various inhibitors than did oxygen reducti.on t 。 a free radical or the very closely related reduction of cytochrome c.

The study of these differences allowed the workers to sketch tentative schemes for the internal electron ~'ransport and to predict that. the various modes of reduction luay occur at different sites. 51

앓뚫끊룹 갚』쏘. I 따 tiation of sulfite autoxidation by biosynthetic dihydro orotate dehydrogenase.

。 '--'‘------‘- ‘g

Cα3)

ζ꾀 쩍·

αL↓34

꿈·톨 짧릎컵 앓￡컬

_Eai~ 볍￡갚""도g융gal，

휠δl

￠찌

L- 싫 φ 영· 형 (s 청 1° 밟 뼈) 때표짧 f1 얹짧 OJ 짧뭘쁨AX때 52

꿇짧앓훌 후요. Initiation of sulfite autoxidation by biosynthetic dihydroorotate dehydrogenase.

The reaction mixture (2 ml) consisted of 200 μmoles of potassium phosphate buffer (pH 7.7) , 2 μmoles of EDTA , 24 pmoles of- sodium dihydroorotate, 100 μmoles of sodium sulfite, 0.2 m! of a l/SO dilution in AQ of a 3,000 unit/ml solution of enzyme (specific activity about 6,000 units/mg of protein) , and (w-here indicated) 300 μmoles of mannitol, 0.5 or 0.1 μmoles of Titan. Control mix tures were identical except that DHO was replaced by water. The reactions were initiated by tip~ing in enzyme and sulfite from separate side arms and the oxygen consumption mea~ured in a Gilson differential respirometer. Symbols: 짧J ， sulfite present; (생) , sulfite + 0.25 roM Tiron present; (口)， sulfite + 0.50 roM Tiron present; (0) , no sulfite present; 짧)， mannitol present. 53

Evidence thus far presented has shmvll that biosynthetic DHO de-

hydrogenase is capable of oxidizing DHO to orotate using as eleGtron

acceptors , oxygen (26) , via a two electron transfer yielding hydrogen

peroxide (Figure 3) or a free radica1 mechanism (Figure ]패4)μ’ va떠u

redox dyes such as potassium ferricyanide (21) ’ p-nitro blue tetrazolium

(26) , and as will be shmvn, cytochrome c and DCI(Table VI). Since the

electrons reducing the various acceptors are tnost likely derived from' at

least two sources (FMN and iron) it is possible that differences in be-

havior with various inhibitors will allow some discussion as to the

scheme of the internal electron transport of biosynthetic DHO dehydro‘

genase o

Table VI shows the effect of p-HMB , a sulfhydryl inhibitor, and

Tiron, a ferric iron chelator, on the reaction rate of biosynthetic DHO

dehydro 당 e.uas 잔 in the prεsence of ferricyanide, DCI , oxygen, or cyto

chrome c as the electron acceptor. The effect of Tiron on sulfite

autoxidation is also shm·nl o All activities tested were found to be in-

hibited by p-Ht~ ， but only cytochrome c reduction and sulfite autoxida-

tion were inhibited by Tiron. The inhibition of ferricyanide reduction

by Tiron could not be measured and the mea딩 urement of the inhibition of

sulfite autoxidation by p-lH1B was complicated by the quenching of the

free radical reacti.on by the organic constituents in the reaction mixtur강。

The inhibition of cytochrome. c by Tiron nlUS t. be interpreted with care.

Miller and Massey (55) have postul쉰 ted that the observed Tiron inhibi-

\ tion may be due, at least in part. to the reoχidation of reduced cyto-

chrome c by Tiron. Hmvever J Aleman and Handler in a later paper (9) did

not mention such a phenomenon \vhen reporting their inhibition data. As 54

TABLE VI

EFFECT OF TIRON AND p-HY.DROXYMERCURIBENZOATE ON DIHYDROOROTATE DEHYDROGENASE ACTIVITY WITH VARIOUS ELECTRON ACCEPTORS - . Inhibitor

앓찮ME T’ iron Acceptor or sub- stance Concentration Concentration 。xidized of inhibitor Inhibition, of inhibitor Inhibition

-쩌1

pM %1 nlM %

FeCN 2.0 75 DCI 2.0 80 1.2 3.3 2.4 3.8 Oxygen 1.0 60 0. {fO 6.0 1.2 -4.0 Cytochrome c 2.0 40 0.33 27 6.0 83 0.66 54 Sulfite 0.25 47 autoxtdation 0.50 72

The reaction mixtures (3 ml) contained 300 μmoles of Tris-HC! buffer (pH 7 e 7) s' 2 pmoles of potass'ium ferricyanide, 0.2 μmoles of DCI , 1 mg of cytochrome c , or no added acceptor, 36 μmoles of sodium dihydroorotate, the specified amounts of inhibitor (not' present in the unit/m1'soll止 ion 'controls) , 0 0 2 ml of a 1/50 dilution in AO of a 3 ,000 of enzyme (specific activity about 6 ,000 units/mg of protein) was used in the cytochrome c assay~ The enzyme was added to the buffer and water followed by given amounts of inhibitor at a specific time. This

mixture (2 0 5 m!) was allowed to incubate 5 minutes and the reaction initiated by the addition of DHO and acceptor (0.5 ml) , DHO and \Vater (0.5 ml) '\vhen 0쩌rgen was used as the acceptor. The experimental condi tions of the measurement of sulfite autoxidation was as stated in Figure 14. Ferricyanide and DCI reduction was followed ,spectrophotometrica11y at 420 nm and 600 nrn respectively, using a blank consisting of buffer,

DRO t and water. Orotate production (acceptor, oxygen) was followed at 282 nrn using a blank containing buffer, enzyme, water, and Tiron equi valent to that to that used in the' reaction mixtures (p~HMB does not absorb at this \vavelength) .. · Cytochrome c reduction was fol1oV7 ed at 550 nm with a blank consisting of buffer, illIO , cytochrome c and ~ater$ Sulfit~ autoxidation was measured as stated in Figure 14. 55 admitted by Miller and ~assey (55) , however, this possible artifact does not explain the observed iW1ibition of sulfite autoxidation·by

Tiron. Since these tw~ processes proceed via the same mechanism (oxy gen free r 큐dical formation) it is· possible that cytochrome c reduction is indeed inhibited by Tiron, but the observed decreases in activity are also partially due to the above mentioned artifact.

These results suggest that the sulfhydryl inhibitor blocks elec~ tron transfer early in the reaction sequence 룰 presumably by a Imechanism

~hich prevents the binding of the substrate, or which interferes with the transfer of electrons from an initial acceptor to one succeeding.

The fact that Tiran only inhibits sulfite autoxidation and (probably) cytochrome c r~duction ， which is mediated by an oxygen free radical bridge between the enzyme and acceptor (54,52) suggests that either the iron groups are reduceq subsequent to the reduction of the flavin, or

Tiron: specifically inhibits oxygen free radical formation, not affect- ing.a possible electron transport function of the iron.

월릎짧앓끓n 0 효 꿇짧잃옳 꿇꿇앓짧n 후잃짧앓꿇릎 힘￡ 앓짧뿔앓짧짧훌옳앓앓앓앓e1많를옳

Table VII allmvs a comparison of the rates of reaction exibited by DHO dehydrogenase using several different electr。낀 acceptors. Under the conditions employed the redox dyes exhibit the most rapid reaction, oxygen and cytochrome c being far less efficient. This general occur-

dehydrog 단 nase renee was also found with catabolic DHO (6 J 55 ,9). 56

TABLE VII

RATE OF OXIDKfION OF DIHYDROOROTATE TO OROTATE BY BIOSYNTHETIC DIHYDROOROTATE DEHYDROGENASE USING VARIOUS ELECT~ON ACCEPTORS

후짧앓짧￡ Reaction rate pmoles DRO oxid/min/ml of enzyme

Ferricyanide 52.5

DCI 75.0

Oxygen 2.5

Cytochrome c 0.89

The experimental conditions were identical to those of Table V except that inhibitor was not present~ Standard curves were prepared to relate change in absorbance to total pIDoles of DHO oxidized in a reaction mixture of 3 mI. 57

DISCUSSτON

Internal Electro민 싫짧짧앓￡

꿇앓둠앓훌￡ 짧앓 밀앓짧짧흘응뿔뚫. 'Figure 15 represents the internal electron transport scheme of catabolic DRO dehydrogenase proposed by

Aleman and Handler (9). Quantitative determinations have shown that

SH 뿔 D 용총 II DHO NAD .. Fe' Fe 4;양II F뻐 (

ι’’’I I ~orot 아샤 e

°2 02 ('02-)

￡후￡끓효용 뚫. Internal electron transport in catabolic dihydrooro tate dehydrogenase.

both Fr-:lN and FAD are present , in the enzyme along \-lith non-heme iron

-때 따 -r 、 돼 빼 -4 뻐, F A 1 때 / 7 Q Q t -4 ’ 따 ---4 h V e S l ‘ . 따 + ” -- 얀 e s L v u A 。 r 、 I Q J , e ι’ a n L m a。 ‘ ,. ,. / . mediate electron carriers as shown by the fact that under anaerobic conditions they were bleached 1 by varying degrees upon the addition of either DHO or NADH. The demonstration of a stable enzyme-orotate complex and obs앙 rved decreases in fluorescenc~ upon addition of orotate characteristic of those occurring when compounds bind to free F~ffi ， suggest that Ftlli is the binding site and the immediate electron donor for orotate. It was predicted that t~e FAD moiety serves as the first

lBleaching of' flavins is an 6ccurrence characteristic of their reduction and consists of extensive decreases in extinction usually measured at the 450 nrn absorption peak.

‘.. 58

electron acceptor for NADH (9). Iron was found to serve as an interme-

diate electron carrier from several lines of evidence including behavior

of flavin-free iron protein derived from the enzyme , ~n electron paramagnetic resonance signal typical- of reduced iron, and the presence of

free radical reactions occurring at-the ir~n site (9). The catabolic enzyme exhibits a marked cysteine activation (5 ,6 ,7,9) and p-chloromer-

curibenzoate (p-CM.B) inhibition (9)· of the activiti.es involving DIlO or

。 rotateo Also, the fact that the NADH oxidas 원 activity is unaffected by these compounds suggests that a sulfhydryl group on the enzyme must be in the reduced condition before the flavin at the DHO-orotate site

·can transfer electrons to or receive electrons from the iron site.

These data also suggest that the FMN at the DHO-orotate site is not

autoxidizableo The fonnation of oxygen-free radicals-has been shown to

occur at the iron residue.

짧짧많강료 P획;과흥뚫. The internal electron transporf scheme proposed by f’ridovich and Handler (49 ,50) is shown in Figure 16 0 Though dif-

-욕 /꺼 °2(·°2-) hypoxanthine 、 Fe~ / oxidized dyes , oxygen \ /\\, ( ) FADl -I FAD2 ( xanthine 와/ NFe/ \~reduced dyes , hydrogen peroxide

M낭 \%2(·O2-)

짧짧￡훌훌용o Internal electron transport in xanthine oxidase.

ferent in some respects , thi.s scheme is quite analogous to that of

catabolic DHO dehydrogenase. The substrate hypoxanthine has been shO\m

to bind specifically to flavin-l from which the electrons are transferred 59 via ferric mεrcaptide groups t 낀 flavin--2 , and from th 낀 I’ e to oxygen or other acceptors. Binding of hypoxanthine to flavin-2 can be induced by the presence of a large excess of the substrate and results in an inhi- bitionof the processes carried out by that-moeity. Excess substrate inhibition Qf the aerobic oxidation of hypoxanthine demonstrated that flavin-I, like theF~~ of catabolic DHO dehydrogena 딛 e ， is not autoxidi- zable. Likewi딩 e cyanide.and p·-CIvrB interfered 'i'lith the abi.lity of- the iron complex to transfer electrons from the first to the second flavin.

The εnzyme was also found to initiaε단 the reduction of oxygen to a fre 단 radicalι

갚훌os짧짧갚짧잃sl깊L있; 갱많잖;갚포짚，찮료끊끓설료. Sine 당 catabolic D깐o d e.hydro원na셀 and xanthin윈 。x i- das€: are similar in several respects it is pODs :i.ble that, in Ii장 ht of t.he properties shown, biosynthetic DHO dehydrog 띤 n 감딩건 may £0110\" a 딩 imilar scheme of internal electron transpor仁 ~ Th낀 u센i ve'rification awaits detailed studi.es of the physical and ~unctional natur깐~ of the biosynthetic enzyme the proposed electron transport scb딩m 윈 of Figure 17 is consistεat with the data present당d in Results. It has

‘ /"_ oxidized dye앉" ox)'설en o ￥}1N ?L당. l Iron --용 FMN f 2 ‘ 、~ redu 앙 ed dyes , hy 년 rag ξn P 쉰 roxide

￡많옳앉표뜸 lL~ Proposed sequ션nee of internal electron transport in biosynthetic dihy건rooT- otic dehydrogεna. se.

been shO\vn directly that biosyntheti.c DIlO dehydrogenase is an FHN-con-‘ taining flavoprotein (Figur단 4) l'‘abIes II and III) and indir안 ct조y that 60

FMN and iron are reduced during the oxidation of DRO to orotate (Figures

3 and 14). A functional sulfhydryl moiety is necessary for activity

(Figures 11 and 12, Table V) and inactivation of the group(s) halts electron transport early in the sequence (Table VI). Hith the evidence thus far obtained, the sulfhydryl group(s) function in one of the following \vays: 1) binding of D표o to the enzyme, 2) electron transfer be t\veen DHO and flavin-I , or 3) the transfer of electrons between flavin-I and the iron c:omplexo It is logical to predict that alternative 3) exists with biosynthetic DRO dehydrogenase by an:alogy with the catabolic enzyme and xanthine oxidase. In these enzymes the sulfhydryl mo i.eties function in the transf딛r of electrons from the primary flavin to the

iron complex~ while substrates bind to the flavin moieties ,.,rithout sul-f hydryl participation. To verify alternative 3) for the biosynthetic enzyme , studies will have to be done to show that with p-HMB inacti vated enzyme DRO still binds to the enzyme and reduces the primary flavin.If the sulfhydryl function is as predicted the results presented in Table VI (the p-HMB inhibition of dye reduction and DHO oxidase activ ity) suggest that flavin-I is not capable of transferring electrons to the terminal acceptors, but rather transfers electrons to an interme diate carrier. It is possible that iron functions as the intermediate carrier, passing electrons from flavin-l to a second flavin from which dyes and oxygen are reduced.

The observation that Tiron specifically inhibits sulfite autoxi dation (Table V工) has also been reported for χanthine oxidase (54).

A possible explanation of this finding may'be that chelation of the iron b)f' T’iron renders the site of free radical formation unavailable

‘.. 61

~o oxygen, sulfite, or ~ytochrome c , but dries not significantly alter

the ability of iron to transfer elec~rons to flavins.

짚매paris으표 요f C꿇짧앓잃 짧양 짧앓짧앓짧흙￡많g 앓앓앓앓앓많융

Table VIII outlines the major physical and kinetic properties

determined for catabolic and biosyntheti~ DRO dehydrogenase. Because of

the availability of only low concentrations of the biosynthetic enzyme

several determinations such aS , flavin, iron and sulfhydryl content have not been made on a quantitative basis. The striking differences be-

tween the tw'o enzymes is in the area of molecular weight, flavin con-

tent, and pyridine nucleotide‘ linked activities. Bi.osynthetic DHO

dehydrogenase has a molecular weight roughly one half that of thecata

‘‘ bolic enzyme and does not contain either FAD or the associated pyridine nucleotide-linked activities•. In contrast to the catabolic enzyme, bio

syntheti~ DRO dehydrogenase does not exhibit stimulation by cysteine or mercaptoetqanol. The two enzymes appear to be similar in all other re spects except perhaps in terms of Km.

It is int당 resting t~ note that in high concentrations of guanidine hydrochloride catabolic DHO dehydrogenase dissociates into four subunits with molecular weights of 30,000 to 31,000 each. Since there are four

'gram atoms of iron per mole of enzyme it was predicted that the enzynle was a tetramer (17)G Since upon dissociation both flavin and iron are released it is to date not possible to determine neither the prosthetic

group content nor how they were attachedG HO~vever it is not unreasonable

to predict that each subunit contains a flavin and an iron moiety ~ tw。

involving FNN and t"70 involving FAD.. In light of 仁he evidence relating 62

TABLE VIrI

PROPERTIES OF CATABOLIC fu~D BIOSYNTHETIC DIRYDROOROTATE DEHYDROGENASE

pν 앞 ，랴 따 --4 페 c Biosynthetic DHO AU ，않 , 織 Property mt m 。。e dehydrogenase

-‘..’ Molecular weight 115 ,000 土 7,000 (9) 53,000 to 57 ,000 (26) 112,000 to 124 ,000 (17)

Number of subunits 4 (17) 2?

?‘ 14않 돼 D qι 뼈 Flavin content m --’ es FMN 뼈 야‘ 。f r mo Ilie 앤、 짧 Vtm l

I4 l 않 따 빼 1* t·4 Sulfhydryl content m ni e o present M 뼈 /l e vi 、 ”

I 함 랴 빡 Iron content 나 am omS present 뼈 /，‘、n 、 f 냥 l e o enzVJm ，/ 뼈 o ·-Ad 샤아 --”&。 tli H O r x m + (6,18) (21,22 ,26) N D돼 1Wt‘ 。 r 獅 .e and o 않 마

Ox ”때+돼t ·m 。 tι Dmω 1bvt

/，‘、 야 D q41 강 ?‘ • ‘, + (6) ’ f O 經 n + e + (6) R ‘때 x 마 않 + t + (9)

Demonstrates oxygen + (9) + free radical forma~ tion

oJ t o o F「 빼 Km for DRO 0.18 mN , acceptor oxygen (8) .J ’ a 랬 Pt 。 r ,‘t 렀 --w an m” e 0.18 mH , acceptor NAD' (9) ‘

Km ,Ki for orotate Km = 2.9 uM , electron Ki = 0.1 mM donor NADH (9) 1.8 mH , electron donor NADH (8)

pH optimum pH 8‘ 2 for ~eduction of pH 7.6 to 7.8 for oxi NAD+ by dihydroorotate dation of DHO by and dihydroorotate ferricyanide oxidase activity

‘ . 63 to the molecular "\veight ,. flavin content, and lack of pyridine nucle。‘ tide-linked activities of biosynthetic DHO dehydrogenase (Table VIII) it is tempting to speculate that the biosynthetic enzyme i.s a dimer , consisting/of FMN and iron-containing subunits, and that the catabolic enzyme is formed by the addition of·two more FAD containing subunits to support.the pyridine nucleotide-linked reduction of orotate to DHO.

However , to date no evidence has been provided to support such an occ·ur·- renee. Studies with mutants 1vould be ?specially valuable in this regard. 64

su빠1ARY

1. Biosynthetic dihydroorotate dehydrogenase was isolated in an ex-

tensively purified form from Lactobacillusbulgaricus•

2. Kinetic studies were carried out including the determination of the

Km for dihydroorotate. Ki for orotate, pH optimum. and the behavior

。 f the enzyme in response to sulfhydryl inhibitors and ammonium

sulfate.

3. The enzyme was found to contain FMN and.indirect evidence shows

that iron is also present. Inhibitor studies have shown that a

group must be in-the reduced sulfhydryl form in order for the enzyme

to function.

4. A possible scheme of internal electron transport for biQsynthetic

기 DHO dehydrogenase is presented.

5. A comparison between catabolic and biosynthetic ’ DHO dehydrogenase

is made on the basis of physical and kinetic propertiese REFERENCES

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37. Stahl, E. , 투부과 L훨똥 Chromatography, Springer-Verlag, New York: 1965. 38. Knight, Jr. , E. and R.W.F. Hardy , ~. 필와• .£뿔땀. 241:2752 (1966). 39. Hinkson, J.W. , and W.A. Bulen, ~. 월와· 탤델. 242:3345 (1967).

40. Massey, V. , G. Palmer, C.H. Williams Jr. , B.E.P. Swoboda , and R.H. Sands in E.C. Slater, (ed) , Flavins 말a Flavoproteins, Elsevier Publishing Co. , London: 1966 , 133.

41. Massey V. , and G. Palmer, Biochem.'5:3181 (1966). 42. Dixon, M. , and E.C. Webb , Enzymes , Academic Press , New York: 1964, 443ff, 117. 43. Friedwa1d, J.S. , and G.D. Maengwyn-Davies in W.D. McElroy and B. Glass, (eds) , 투뿜 Mechanism 앞 Enzyme Action, Johns Hopkins Press, Maryland: 1954, 157.

44. Webb , J.L. , Enzyme 뿜휴 Metabolic Inhibitors, Vol. I , Academic Press, New York: 1963, 55ff, 151, 552ff.

45. Whittenberger, C.L. , and A.S. Haaf, Biochemica 뚱 Biophys. 소뜩!' 122:393 (1966).

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48. E’ridovich, I. , and P. Handler, 노· 월랴· 댈ξ믿.， 228:67 (1957).

49. E’ridovich, I. , and P. Handler, ~. 혹굉추. C뿔무.， 231:899(1958).

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51. E’ridovich, I. , and P. Handler, 노· 받와· 않댈.， 236:1836 (1961).

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55. Miller, R.W. , and V. Massey, I. 봐않• .9길웰" 240:1466 (1965). APPENDIX

DERIVATION OF THE RELATIONS USED IN DETERMINING THE Km FOR DIHYDROOROTATE AND A METHOD FOR DETERMINING Ki FOR OROTATE

The following derivation is based upon equilibrium kinetics where it is assumed that an inhibitor can either affect the affinity of the enzyme for its substrate or change the rate at which the enzyme-substrate complex breaks down into enzyme and products (44, p.56). Such a relationship is shown in equation 1:

彩/갖 \췄칙

α~ EI + P (Eq. .1)

Where: E = enzyme, S = substrate, P = product, I = inhibitor, ES = enzyme-substrate complex, EI = enzyme-inhibitor complex, EIS = enzyme-inhibitor-substrate complex, and: a = change in the affinity caused by the inhibitor

a = inhibitor induced change in the rate of complex decomposition.

κs 혹 = dissociation constant of the ES complex (E)(S) CEq. • 2) (analogous to Km) • (ES)

Ki 톨 dissociation constant of the EI complex = (E)''""or、(I) (Eq. 3)

률 aKs = • dissociation constant of the EIS complex (El) (S) (Eq. 4) (EIS) 69

Y Ki 톨 dissociation constant of the EIS complex = (ES) (I) CEq. 5) (EIS)

During an inhibited enzymatic reaction the total enzyme concentra-

tion is represented by Equation 6:

(Et) 혹 (E) + CES) + (EI) + (EIS) (Eq. 6) and"the rate of product formationby Equation 7:

[뿔)i 률 Vi = k(ES) + SkeElS) (Eq. 7)

So that Equation 6 may be expressed entirely in terms of the ES complex,

Equations 2 to 5 are rearranged and substituted as follows:

Ks = (E)(S) ~‘ (E) = (ES) 스동 「 (Eq. 8) (ES) (S) l f -→~ 얻동 「 i = (E) (I) (EI) = (E) (I) = (ES) (I) = (ES) (I) (Ks) (Eq. 9) (E11 ---r ,--, Ki Ki (S) "~UI (~). Ki: 1 'f s = (E1)(S) -+ (ElS) 닐 (EI) (S) = (ES)(I)짧 4월 = (ES) 와j (EIS) αKs 짧~Ki Ct~’ α Ki (Eq. 10)

i = (ES) (I) ‘ -+ (EIS) _(ES)(l) = (ES) ~다 (EIS) , ,---, YKi

The constants ct and yare found to be equal by Equating equations 10

and 11:

(ES)싫 톨 (ES) 싫 :. ex 흩 Y

Summarizing: (E) 률 (ES)얻혹 CEq. 8) (S) 70 떠”……쩨*때 째써 야 /tl ω 、 i/l (Eq. 9) ‘、 …#때 때 야m (Eq. 12)

Substituting·Equation 8 , 9 , and 12 into Equation 6 yields:

Et = (ES) 혈j + l + {접 織 + &짧 (Eq. 13)

Rearranging:

Et (ES) 흐 혈 + 1 +파앨흑+일L (5) - (S) Ki aKi

Eta(S)Ki = (Eq. 14) 어(iKs + a(S)Ki + a(I) d(s + (S) (I)

Substituting Equation 12 for (ES) by Equation 14:

(5) (1)1 aKi

Et(S)(I) (Eq. 15) a [KiKs + (S)Ki + (I)Ks] + (S) (I) L

Substituting relations 14 and 15 into Equations 7 yields:

Vi ::I k Etα (S)Ki α 단(iKs + (S)Ki + (I)K략 + (5)(1)

8 k· Etα (S)Ki aC진il

~~k(S)(Et~a S(I 끼 g Ki + (Eq. 16) ’ ‘ q 다iK s + (S)Ki + (I)K 박 + (5)(1) 71~~

If an uninhibited enzyme reaction is allowed to proceedat saturating substrate concentrations then essentially all of the enzyme is in the form of the enzyme-substrate complex. Under these conditions Vi = k (ES) may be written as Vmax = k(E 다 , where Vmaχ is the'reaction rate at saturating substrate concentrations. Applying this assumption t 。

~~Equation 16 yields the general rate equation:~~

~~Vi 톨 (Eq. 17) (8) (I)~~

~~Equation 17 may be written in the Michaelis form:~~

타까 와 alxl α- 없-++-n-+-+Qμ--glf딩 /Il야 찌 l/ L-+ ‘‘、-- 、 --Ka·s -/ll-야 -「따띠 -와 톡 Vi .:: l --n-) (8) Vp1ax’ 없-떼 (8) + KIn’ n 、 r ‘ where the observed maximum velocity, Vmax ’ = Vmax αKi -+:6 (I) (Eq. 18) 야Ki + (I) and the observed KIn. Km' = Ks αKi + et(I) (Eq. 19) ’ αKi + (I)

Equation 19 shows how the observed Km (Km') of an enzymatic reaction varies with inhibitor concentration. If the inhibitor exhibits com- pletely competitive inhibition, then in relation to Equation 1 and 19 a a 00.

~~Applying this value of a to Equation 19 (rewritten in Equation 20):~~

~~Km' (Eq. 20)~~

~~as a ~ GO , 1/a --;... 0 and Equation 20 becomes: m* Ki + (I) 떼 Km' 톨 K8 = Ks 톨 Ks 봐 (I) + 1 (Eq. 21) Ki l\.~ 72~~

It can be seen from Equation 21 that if one plots Km'· vs (I) a straight line will result from which Ks may be obtained from the ordinate intercept at (I) - 0 and Ki may be determined from the slope.

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