Portland State University PDXScholar 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 Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Let us know how access to this document benefits ou.y 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