Severo Ochoa's Contributions to the Citric Acid Cycle
Year: 2005
Type: article
Abstract: Enzymatic Synthesis of Citric Acid. I. Synthesis with Soluble Enzymes(Stern, J. R., and Ochoa, S. (1951) J. Biol. Chem. 191,161-172) Enzymatic Synthesis of Citric Acid. IV. Pyruvate as Acetyl Donor(Korkes, S., del Campillo, A., Gunsalus, I. C., and Ochoa, S. (1951) J.Biol. Chem. 193,721-735) Severo Ochoa (1905-1993) was born in Luarca, Spain and became interested inbiology while in high school. Because there were few graduate programs in thebiological sciences in Spain at that time, he had to enroll in the MedicalSchool at the University of Madrid to study biology. Fortunately, during hissecond year, Ochoa was offered the opportunity to do research. His firstexperiment, isolating creatinine from urine, stimulated his interest in thefunction and metabolism of creatine and creatinine in the body. As a result,he and José G. Valdecasas came up with a simple micromethod for thedetermination of creatine in muscle. They submitted a paper on this method tothe Journal of Biological Chemistry (JBC), and to their delight, itwas published in 1929 (1Ochoa S. Valdecasas J.G. A micromethodfor the estimation of total creatinine in muscle.J. Biol.Chem. 1929; 81: 351-357Abstract Full Text PDF Google Scholar). SaidOchoa, “Not even in my wildest dreams could I have dreamt that, yearslater, I would become a member of the editorial board of the JBC and serve asPresident of the American Society of Biological Chemists (now the AmericanSociety for Biochemistry and Molecular Biology)”(2Ochoa S. The pursuit of a hobby.Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Scopus (20) Google Scholar). 1All biographical information on Severo Ochoa was taken from Refs.2Ochoa S. The pursuit of a hobby.Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Scopus (20) Google Scholar and7Kornberg A. Remembering our teachers.J. Biol. Chem. 2001; 276: 3-11Abstract Full Text Full Text PDF PubMed Google Scholar.1All biographical information on Severo Ochoa was taken from Refs.2Ochoa S. The pursuit of a hobby.Annu. Rev. Biochem. 1980; 49: 1-30Crossref PubMed Scopus (20) Google Scholar and7Kornberg A. Remembering our teachers.J. Biol. Chem. 2001; 276: 3-11Abstract Full Text Full Text PDF PubMed Google Scholar. After completing his medical degree in 1929, Ochoa spent the next 13 yearsin a total of 12 different laboratories (9 in Europe and 3 in the UnitedStates), partly because he sought out laboratories at the frontier ofphysiology and medicine but mostly because of social and political turbulencein Europe. Ochoa's first stop was the Kaiser Wilhelm Institute for Biology inBerlin-Dahlem where he worked with Otto Meyerhof, Nobel laureate and author ofa previous JBC Classic (3Meyerhof O. Junowicz-Kocholaty R. J. Biol. Chem. 1943; 149: 71-92Abstract Full Text PDF Google ScholarMeyerhof O. J. Biol. Chem. 1945; 157: 105-120Abstract Full Text PDF Google ScholarMeyerhof O. Oesper P. J. Biol. Chem. 1947; 170 (JBC Classics, (http://www.jbc.org/cgi/content/full/280/4/e3)): 1-22Abstract Full Text PDF Google Scholar), onthe chemistry of muscle contraction. In 1932, Ochoa moved on to the NationalInstitute for Medical Research in London to work with Harold W. Dudley onglyoxalase and Sir Henry Dale on the influence of the adrenal glands on thechemistry of muscular contraction. He then returned to Madrid where he studiedglycolysis in heart muscle. Unfortunately, in 1936 Ochoa's work came to a standstill with the SpanishCivil War, and it became apparent to him that he would have to leave thecountry if he wanted to continue doing research. With that in mind, hereturned to Heidelberg to work with Meyerhof on some of the enzymatic steps ofglycolysis and fermentation. However, with Adolf Hitler's rise to powerMeyerhof left for Paris, and Ochoa went to work with Archibald Vivian Hill atthe Marine Biological Laboratory in Plymouth, England, and later R. A. Petersat Oxford University. At Oxford, Ochoa began working on oxidative phosphorylation and showed thatoxidation is coupled to phosphorylation of AMP to ATP followed by a transferof phosphate from ATP to the sugar. Ochoa's research was again cut short bywar, this time World War II, and he left for the Washington University Schoolof Medicine in St. Louis to work with Carl and Gerty Cori, future NobelLaureates and authors of a previous JBC Classic(4Cori C.F. Cori G.T. J. Biol. Chem. 1928; 79: 321-341Abstract Full Text PDF Google ScholarCori G.T. Colowick S.P. Cori C.F. J. Biol. Chem. 1938; 124: 543-555Abstract Full Text PDF Google ScholarCori G.T. Colowick S.P. Cori C.F. J. Biol. Chem. 1939; 127: 771-782Abstract Full Text PDF Google ScholarGreen A.A. Cori G.T. J. Biol. Chem. 1943; 151: 21-29Abstract Full Text PDF Google ScholarCori G.T. Green A.A. J. Biol. Chem. 1943; 151 (JBC Classics(http://www.jbc.org/cgi/content/full/277/29/e18)): 31-38Abstract Full Text PDF Google Scholar). After spending 2 yearswith the Coris working on the enzymatic mechanism of conversion of fructose toglucose in liver extracts, Ochoa finally acquired a permanent faculty positionat New York University (NYU) and remained there until his retirement. At NYU Ochoa resumed his studies on oxidative phosphorylation, this timeconcentrating on the enzymatic reactions that were coupled to phosphorylation.Hans Krebs had shown that the citric acid cycle was the main oxidative pathwayfor food in the cell, and David Keilin, Otto Warburg, and others haddiscovered many of the enzymes that were involved in transferring hydrogensand electrons from oxidizable substrates to molecular oxygen. However, verylittle work had been done on the enzymes involved in the primary oxidation ofcitric acid cycle substrates. Ochoa decided to focus on these enzymes,including what he termed was “the most elusive enzyme of the citric acidcycle,” the enzyme that condensed oxalacetate with “activeacetate” to form citrate in the first reaction of the cycle.Acetyl-CoA+oxalacetate+H2O→citrate+CoA+H+REACTION 1 Ochoa believed that this “condensing enzyme” was soluble butthat the enzyme(s) that made “active acetate” (acetyl-CoA) wereinsoluble, at least in animal tissues. Thinking the enzymes might, however, besoluble in bacteria, Ochoa and his postdoc Joe Stern combined anEscherichia coli extract with an extract of pigeon liver, asdescribed in the first JBC Classic reprinted here. Ochoa and Stern reportedthat their pigeon liver extract could catalyze the synthesis of citrate fromacetate, ATP, and oxalacetate if coenzyme A and magnesium were added. Whenthese extracts, or other types of animal extracts, were complimented with“acetate-activating” E. coli extracts (which bythemselves may or may not synthesize citrate) in the presence of oxalacetate,citric acid was formed. Ochoa and Stern concluded that the E. coliextract provided transacetylase (an enzyme discovered by Earl Stadtman), whichcatalyzed the transfer of phosphate from acetyl phosphate to CoA to formacetyl-CoA and phosphate. Using this reaction as an assay, Ochoa waseventually able to crystallize the condensing enzyme from pig heart(5Ochoa S. Stern J.R. Schneider M.C. Enzymatic synthesis of citric acid. II. Crystalline condensing enzyme.J. Biol. Chem. 1951; 193: 691-702Abstract Full Text PDF PubMed Google Scholar) and show that it catalyzedthe reversible conversion of acetyl-CoA and oxalacetate to CoA and citrate(6Stern J.R. Ochoa S. Lynen F. Enzymatic synthesis of citric acid. V. Reaction of acetyl coenzyme A..J. Biol. Chem. 1952; 198: 313-321Abstract Full Text PDF PubMed Google Scholar). Ochoa was also interested in the early steps of pyruvate oxidation and themechanism by which pyruvate and oxalacetate reacted to form citrate. In thesecond JBC Classic, I. C. Gunsalus, who was spending a few months in Ochoa'slaboratory on a Guggenheim Fellowship, and Seymour Korkes isolated thepyruvate oxidation system from E. coli in two fractions (A and B).They found that in the presence of diphosphopyridine nucleotide (DPN, nowcalled nicotinamide adenine dinucleotide or NAD) and CoA, their fractionscatalyzed the conversion of pyruvate to acetyl phosphate or to citrate whencondensing enzyme and oxalacetate were added. They concluded that theirfractions catalyzed the following reaction to produce acetyl-CoA from pyruvate.Pyruvate+CoA+NAD+→acetyl-CoA+CO2+NADH+H+REACTION 2 Although Ochoa's contributions to the citric acid cycle were significant,they were eclipsed by a chance finding he made in 1955 that would win him theNobel Prize 4 years later. He and his postdoc Marianne Grunberg-Managodiscovered an enzyme that converted ADP and other nucleoside diphosphates intoRNA-like (NMP)n polymers. Initially they hoped thisenzyme, named polynucleotide phosphorylase, might be responsible for thebiosynthesis of RNA. However, the realization that polynucleotidephosphorylase did not need a DNA template for RNA synthesis dispelled thisidea. This initial disappointment was soon forgotten when Ochoa realized thathe could use polynucleotide phosphorylase to synthesize a variety of RNA-likepolymers and identify many of the nucleotide triplets that encoded for aminoacids. In 1959 he was awarded the Nobel Prize in Physiology or Medicine withArthur Kornberg, one of his first postdocs at NYU, “for their discoveryof the mechanisms in the biological synthesis of ribonucleic acid anddeoxyribonucleic acid.” It was subsequently determined that the functionof polynucleotide phosphorylase was to degrade RNA, not synthesize it, despitethe fact that the enzyme runs its natural reaction in reverse invitro.
Source: Journal of Biological Chemistry
Language: English
Subfield: History and Philosophy of Science
Field: Arts and Humanities
Domain: Social Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1585359102