How I became a biochemist, twice

Background: Born in Chile in 1939, my childhood was deeply influenced by the Second World War, the Holocaust, the ideologies that flourished at the time, the proclamation of the State of Israel, and the beginning of the Cold War. Chile, a country where the mountains and the sea are permanent references, induces decisions based on firm and clear landmarks. My family was large and passionate. One grandfather, an intellectual and conservative Jew, ran from the communists after 1922. He was never fully at ease with Chilean society; with him the themes were religion and world politics. My other grandfather left Russia before the First World War and was as integrated in Chilean society as his Spanish pronunciation permitted. He was always ready for good scotch and friendly gossip. Of course my grandfathers did not like each other. My father was a pharmaceutical chemist that enjoyed life with a superlative enthusiasm. My mother, a devotee of all things literary and a Master Chef, later became a writer. One of her last novel's title, “Para Siempre en mi Memoria”1, could represent these reminiscences. One of my uncles, a physician that did some clinical research, was an accomplished dancer and piano player and knew everything about nightclubs. Mother's sister was a communist until her premature death. Saturday family lunches at my parents' home were lively events, with superb food and exciting political debates. Family influences, contradictory as they were, accustomed me to see various interpretations of single events and introduced me to dialectics. The end of the family lunches, with the advent of the Pinochet's dictatorship, reflected the roots of the divide in Chilean society, some of which remain to this day. School: I learned to read and write in English in a private school and completed my education in the Instituto Nacional (Instituto), a public institution where many Chilean leaders have studied over almost two centuries. In high school I was busy partying, dating, skiing, and reading and did not study much, except Chemistry, Biology, Physics, and Philosophy. At the Instituto, we began to learn the theory and practice of politics, which I have been involved in since. In my father's pharmaceutical laboratory I had my first contact with Chemistry. One of the lab consultants gave me chemistry books for children as birthday gifts, so I started experiments very early. Some experiments did not go as planned, and I almost destroyed the family garage. A microscope, fermented leftovers, and garden bugs introduced me to hands-on Biology. With my mother's advice, I read “the Russians” and a number of the classics before I left school. Dr. Arrowsmith was not read without consequences and, never modest, I dreamed on saving humanity through Chemistry and Biology. University as an undergraduate: At age 17, I was admitted to the Facultad de Farmacia of the Universidad de Chile (FF/UCh) to study Pharmacy. During the first semester, I had to stay in bed for almost one month. The social worker that visited me at the end of the rest period advised me to study only one or two disciplines that year. Not liking the idea, I passed all exams and won some prizes as the best student in my class. Taking (some of) the courses seriously made me interested in the basic aspects of Science. The pharmaceutical parts, and my father's lab for the matter, were not attractive for me. At the end of the first year I was getting ready to enter a new selection process for admittance in the Medical School, for I found most of the courses boring. At that time, 50 years ago this year (2007), the late Osvaldo Cori, a medical doctor trained as a biochemist by Fritz Lipmann and Severo Ochoa in the US, created a new undergraduate curriculum: Bioquímica (Biochemistry) at the FF/UCh. Bioquímica attracted a small number of students who were promised to become scientists and make a contribution for the improvement of Science in Chile. The leadership and intellectual density of Osvaldo converted a group of 10 Chilean undergraduates, 18–20 years old, into apprentice scientists in a Professional School. I abandoned Pharmacy, my plans for Medical School and joined the adventure. It was very hard to explain to my father that his plans for me, at his lab, would have to be shelved, or that at least he had to wait until, in his view, I became convinced that being a scientist in Chile was not a way to earn a living. We were so sure we would become the next Ochoas or Lipmanns that nothing else mattered. By creating Science in Chile, we would also solve many of the societal problems of our time. The feeling was exhilarating and some of the other Professors at the time, such as Mario Luxoro, helped to transform the apprentices into (probably insufferable) youngsters convinced that they would change the world through Science. Those 5 years, in particular the last two, were incredibly rich, as our small group received tutoring of exciting Science, as opposed to formal lectures for a large audience. A subgroup, formed by Yedy Israel, Romilio Espejo and myself, studied and worked together, competed and developed a life-lasting friendship. To this day I tell my students that being a scientist, especially in an underdeveloped country, means that you have to believe that your contributions may take you to win the Nobel Prize, and that you must also have the internal fortitude not to get there. In our 4th year, Romilio and I went to work in J. Tohá's lab in the Faculty of Engineering. Our project involved measuring the kinetics of [32P]Pi incorporation into proteins and ribonucleic acids of living cells, an important subject at that time. We enjoyed tremendously working in the lab and were fascinated by the variety of interpretations that could accommodate our data. We had to study a lot in order to understand what Tohá was saying about virus infection, RNA synthesis, and other mysterious-sounding terms. Romilio stayed with nucleic acids and I with kinetics. At the end of that year I was invited by Osvaldo's to work with him as an undergraduate teaching assistant. My first attempt at understanding life was trying to determine the nature of phosphagens in a gastropode's muscle by [32P]Pi labeling using abalones (loco, in Chile). My experience with locos until then had been essentially gastronomical, since this is one of the most sought-after sea foods. Having no experience at popularizing Science, it was quite difficult to explain this research theme to my family. The shift from phosphagens in locos to enzymes in potatoes resulted from a grant refusal. Now I had to explain my work with another food ingredient at family gatherings. Purifying apyrase from potato was an intricate experience involving cyanide-containing buffers to avoid oxidation of the extract. Creativity was forced upon me out of fear to continue to use cyanide-containing buffers in the cold room, so we developed an alternative purification method. With the purified enzyme and my previous experience with [32P]Pi, we used labeled substrates to probe apyrase kinetics. Obtaining pure β and γ [32P] labeled ATP, a project in itself, was an enzyme/column trip. After confirming the purity of AMP-[32P] –P and ADP-[32P], we described apyrase catalyzed hydrolysis in detail. The first article on apyrase I co-authored is still cited2. Working in this lab was an enriching cultural experience. The daily luncheon seminars, in which we discussed results and published work, also included an exposure to Osvaldo's vast culture and political ideas. I enjoyed the Science and the cultural parts. My fascination with kinetics as a tool, enzymology as a subject, Chemistry in Biology and modeling, originated there. I received my degree in July 1962, becoming a Biochemist, at least from the point of view of the Title, at the age of 22. I was the second Biochemist to graduate, a month or so after Yedy Israel left Chile to do graduate work in Canada. After graduation, I was hired by the FF/UCh as an Assistant. During my lectures, Osvaldo and his assistants would sit in the first row and throw tough questions. This experience was difficult but well taken. I had to prepare my lectures carefully, looking, at the same time, for didactic consistency and up-to-date scientific background. I was being educated to teach, formulate tough questions, and to stand to their challenge. At that time C.A. Bunton started visiting Chile regularly on an exchange program. Meeting Professor Bunton (it took me years to call him Bunny) was a life-changing experience. My description of Bunny is published in an Article that I co-authored with two colleagues when the scientific community celebrated his 80th birthday3. University of California at Santa Barbara (UCSB): Convinced that I had to learn more Chemistry to understand enzymes, I went to Bunton's lab in UCSB to do research in Physical Organic Chemistry supported by a Rockefeller Scholarship. I got married to a Brazilian girl before departing to the USA, not realizing, at the time, that this decision would later take me to Brazil for most of my life. I did not hold a PhD, and accepting Osvaldo's advice not to enter graduate school, I went to the US as a visiting scientist. My students have a hard time understanding why, upon arriving in Santa Barbara, I had to build a temperature-controlled oil bath to start studying pyrophosphate hydrolysis. This was a minor task, coming from an advisor that had built a mass spectrometer from scratch. Using “my” bath and a Klett colorimeter, we soon described selective solvation in pyrophosphate reactions in mixed solvents. The period I spent in UCSB was intense. T.C. Bruice's course on Bioorganic Mechanisms covered the ground that was to appear as a book co-authored with S.J. Bencovic. One of my most significant memories of that time is the capacity of Bunny to put an isolated result in global chemical perspective. The revival of micellar catalysis started after a discussion in an evening seminar where I advocated the use of micelles as enzyme models. One of the post-docs at that time, Janos H. Fendler, a distinguished chemist and dear friend who passed away recently, insisted that I should get a PhD, and he was right. Discussing Chemistry, among other themes, over cognac with Janos at his apartment in Isla Vista was one of the joys of Santa Barbara. Harvard: Because I believed I understood phosphate ester hydrolysis by then, I left UCSB to join FH Westheimer's group at the Chemistry Department of Harvard as a post-doc. I had the support of Bunny and a new Rockefeller Scholarship. The change from the Universidad de Chile to UCSB was simple when compared with the UCSB/Harvard transition. It took a lot of very hard work, and some pain, to survive in that Chemistry Department. My first project, dealing with chymotrypsin labeling using phenyl diazoacetate failed and, having no previous experience in organic synthesis, obtaining diazoacetic acid derivatives was a nightmare. It took me some time to feel that I belonged there. At the end of my Harvard year, however, having presented a Departmental Seminar and with a article on models for active site labeling, I felt I had made it. It was an exciting time, with phosphate pseudorotation being discussed most of the time, insights of enzyme catalysis and highly sophisticated scientific thought presented by F.H. Westheimer during seminars. With some friends made at that time, we still remember the intellectual joy of being there. Going Back: I had no doubt that I had to return to Chile after my USA training. I still think that scientists in developing countries have a social responsibility over and above our natural and personal passion to discover, and being recognized for that. Science has not moved Latin America as fast as I then wished for. However, I can also attest the importance of the collective work of scientists for our societies. Chilean Interlude: Back in Chile, again in Cori's lab, a re-entry grant from the Rockefeller Foundation made my scientific independence possible. I still wonder how so many different research lines co-existed in a lab that had an area as large (or small) as my present workplace. Frank Marcus was working with F.D. Pase, Osvaldo was starting his work on terpene biosynthesis. Aida Traverso-Cori continued with potato apyrase while Yedy Israel, back from Canada, worried about ethanol effects on membrane ATPases. Collaboration was restricted to intellectual lunch time discussions and nobody, except the boss, could work with more than one student or technician. I started to work on low molecular weight acid phosphatase, now known to belong to the group of phosphotyrosyl protein phosphatases. My intention was to understand catalysis by an enzyme that increased the rate of hydrolysis of a phosphomonoester, a well understood reaction (or so I thought), without cofactors. At the same time I started to devise models for the nonenzymatic catalysis of the same reaction. Having ascertained my independence from Osvaldo I started, on the side, a fruitful collaboration with Jorge and Catherine Allende by applying steady state kinetics to analyze the threonyl-tRNA synthase catalyzed reaction. My first presentation at an international meeting, with ping-pong kinetics and all that was on the activating enzyme. I cannot remember why I decided to purify acid phosphatase from bovine brain. Possibly it was as far away from potatoes or pine needles (Osvaldo's tissues of preference) as I could imagine. It is also possible that this decision was based on the price of the starting material and contacts in the local abattoir. Purification of the enzyme was easy, after all a low molecular weight (13 kD) acid stable enzyme was a good starting point. The difficulties started afterwards when, in the pre- “express in E. coli” days, we recognized that the final yield of the pure enzyme was a few μg per kilo of brain. We described the purification of the enzyme and some initial kinetic and inhibition data soon afterwards. It was clear, from the beginning, that an active site cysteine was essential for catalysis. Trying to understand the role of the thiol was going to be a major component in my scientific interests. Yedy Israel and myself listened to Osvaldo's advice suggesting that the way to advance in the Chilean academic ladder was to become a “Professor Extraordinario,” a doubtful translation of the German “Privatdozent.” We spent quite some time preparing for a complex series of exams that covered all of Biochemistry and required a Thesis. Going through Biochemistry gave me a solid culture that was instrumental during the course of my career. We were both approved with honors and, in spite of the titles, left Chile in the following months. My marriage was one of the main reasons to leave Chile. Even before returning to Chile from the USA my in-laws had investigated the possibilities for getting an academic position in Brazil and I was already receiving offers. The feeling of suffocation in a crowded lab, where I could see a very little chance to set up a larger research group, weighted as well. University of São Paulo: Thus, with a year of unpaid leave of absence, I arrived in São Paulo at the Department of Physiology in the School of Medicine of the University of São Paulo (USP) with a scholarship from the State of São Paulo Foundation for Research (FAPESP). Despite the somber atmosphere in the Department, caused by the politically driven expulsion of the Departmental Chair, I was received with open arms. I started to recruit students, since FAPESP had also included setting up a lab in the package. Soon afterwards I had a small team of undergraduates, a full time technician and a graduate student purifying acid phosphatase and threonyl tRNA synthase. I felt isolated in the Department of Physiology, despite the incredible support that I was given. My transfer to the Institute of Chemistry (IQ) was negotiated and I arrived to the newly created Department of Biochemistry at the IQUSP. My colleagues at the Department of Physiology generously agreed not only with my transfer, but I was able to take with me the recently acquired Sorvall centrifuge, pH meter, and lab-ware. I re-started (minus the technician) in a small lab that had been left temporarily by a faculty member doing postdoctoral research abroad. This colleague, J.C.C. Maia, who died prematurely, did not like the idea of returning to an occupied space. We started with an acute conflict a relation that developed into a deep friendship. This reference is important when describing how I became a Biochemist, since Maia was a biologist, and my chemically driven Biochemistry was enriched while trying to explain my research to Maia, because he gently led me to try to look at the same problem thinking in terms of Biology. Perhaps if I had listened more, my group would have discovered that the biological substrate of acid phosphatase was phosphotyrosine and my history would have been different. Instead we suggested, based on classical inhibition and kinetic data, that cysteine was the nucleophile and that the covalent intermediate of acid phosphatase contained an SP bond. The atmosphere at the Biochemistry Department of IQUSP in the early 70's was scientifically exciting, despite the dismal political atmosphere in a country run by a military dictatorship. FAPESP had initiated a Program to support Biochemistry in the State of São Paulo (BioqFAPESP) overseen by an international Advisory Committee and a local supervising group which I was nominated to be part of. The legitimacy and scientific significance of FAPESP and the international advisory committee made a deep impact on the Department. Having a Project approved in such a Program gave a number of very young Faculty members, me included, long term funding and independence from the formal university structure. Some people still remember with awe the day when a project presented by a full professor was not approved. Most of the faculty in Brazil failed to understand the impact of BioqFAPESP in the very formal and conservative structure of a Latin American university at the time. To the young faculty members, however, who had been exposed to science abroad, it was natural that space (be it scientific, political or measured in square meters) should be related to the quality of the Science rather than the formal position in the academic ladder. With adequate funding and a small research group already producing results, I had to decide whether to stay in Brazil or go back to Chile, where I had a permanent position. a like I my and, in spite a position in that had to be years, I decided to to my faculty position in Chile. Soon after arriving at I a graduate course in I spent the part of a year preparing the trying to all I knew about kinetics and in a one about in a different basic with the last article published in the was an enriching experience. 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In I an on in São Paulo and, the of coming to Brazil attracted some the best scientists in the The fascination with the basic and aspects of and was soon to the major of research in my Soon I was in the global that gave to a part of what is known as and our contributions to the subject were for the of reaction in the description of formed by fully phosphate derivatives and catalysis of ester by In I was for an active research group, had been the advisor of and was the of the Program in Biochemistry at IQUSP. many that year the was made through a to academic My degree in Biochemistry was not recognized in I did not hold a degree and was no Brazilian for my of by the Universidad de Chile. a the of my by and was to myself and the to to my I started for a position it is difficult for me to remember or understand the reasons my decision to remain in the since I had in abroad, in and A set of very graduate students had joined my group and the atmosphere in the lab was We were obtaining a lot of data, and At some I decided to stay in the but in order to do so I had to a of the University I discovered an article that scientists with to a degree by a Thesis. I do not have the space to the It is to that the IQUSP Faculty on my by a small I presented a my and after in a process that included another and I the of translation of see The results presented in were published and one of the is still one of the most used to analyze reactions in in fast with At with a permanent in the Department of I had become a Biochemist,

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