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 of the was to be a in my scientific Yedy Israel and to Osvaldo's advice that the way to in the Chilean was to become a a of the We spent quite some time for a of exams that covered all of and a Going through gave me a culture that was during the course of my We were with and, in of the left Chile in the My was one of the to Chile. before to Chile from the USA my had the for getting an in Brazil and I was The feeling of in a lab, where I could see a very to a research as University of with a year of of I in at the Department of in the School of of the University of with a from the State of Foundation for the in the by the of the Departmental I was received with I started to since had also included a lab in the afterwards I had a small of undergraduates, a time and a graduate student acid phosphatase and I felt isolated in the Department of the support that I was My to the of Chemistry was and I to the created Department of at the My colleagues at the Department of not only with my but I was to take with me the and I the in a small lab that had been left by a research This who did not like the of to an We started with an a that developed into a friendship. This is important when how I became a Biochemist, since was a and my was while trying to explain my research to he me to to at the same in of Biology. I had my group would have that the of acid phosphatase was and my would have been we based on inhibition and kinetic that cysteine was the and that the of acid phosphatase an The at the Department of in the was the political in a country by a had a to support in the State of by an international and a local group which I was to be The and scientific of and the international made a on the Department. Having a in such a gave a number of very Faculty me and independence from the formal Some still remember with the day when a project presented by a was not of the in Brazil failed to understand the of in the very formal and conservative of a Latin at the time. To the however, who had been to it was natural that it political or in should be to the of the Science than the formal in the With and a small research group I had to to stay in Brazil or go back to Chile, where I had a permanent a like I my and, in a in that had to be years, I decided to to my in Chile. after arriving at I a graduate course in I spent the of a year 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. I about the of the I still the to the students that had to through the Science and put with my since Spanish and do not at My and I that course for a It is possible that in the the about the of a to in became a research I cannot but to with that it took me almost years to solve the After to a simple to the of acid phosphatase in I decided to for another I back to my lab the study of micelles to the of on chemical in In I an international and course on Physical Organic Chemistry with C.A. Bunton as invited One of the themes was and on the of the I co-authored my first article on was an exciting subject and many scientists thought that the way to in fully had been The of models for the of the kinetics was our first and my research group acid phosphatase and micellar as In I an on in 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 of research in my I was in the global that gave to a 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 had been the advisor of and was the of the in at many that year the was made through a to My degree in 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 and the to to my I started for a it is difficult for me to remember or understand the my decision to remain in the since I had in in and A of very graduate students had joined my group and the in the lab was We were obtaining a lot of and At some I decided to stay in the but in order to do so I had to a of the University I an article that scientists with to a degree by a I do not have the to the It is to that the Faculty on my by a small I presented a my and after in a process that included another and I the of 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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