Enzyme Ingenuity in Biological Oxidations: a Trail Leading to Cytochrome P450

Those unfamiliar with basic research in biochemistry and related fields may assume that important discoveries are the result of brilliant ideas that are single mindedly pursued until, many years later, the answer is obtained, perhaps along with important biomedical applications. The progress of science is almost always more haphazard, as ambitious young scientists are influenced by their teachers, by the cooperative or competitive work of others, the availability of new techniques, and chance findings that may lead to different goals. Sixty years ago as an undergraduate at the University of Colorado, I took my first biochemistry course in the Chemistry Department taught by Professor Reuben Gustavson and had the good fortune to be invited by him to join his small research group studying steroid hormones. I had a tremendous amount to learn but was fascinated from then on with research and the possibility of making new discoveries. In these reflections the influence of my mentors/teachers, whom I much admired for their personal qualities and achievements, is acknowledged. Although my research over the years has taken many unexpected turns, a common thread has been an interest in biological oxidations, particularly those not readily explainable according to the predictions of organic chemistry. This curiosity has led to fundamental studies on the properties and mechanism of action of cytochrome P450, now often described as the most versatile biological catalyst known. Although this was not my original goal, the mammalian isoforms of this enzyme have turned out to be of biomedical importance because of their central involvement in the metabolism of steroids, drugs, and chemical carcinogens. William Cumming Rose was a dedicated and inspiring teacher and an outstanding pioneer in biochemistry and nutritional science who spent most of his career at the University of Illinois (1Carter H.E. Coon M.J. William Cumming Rose, a biographical memoir..Biogr. Mem. Natl. Acad. Sci. 1995; 68: 3-21Google Scholar). Young Will attended schools in small communities in North Carolina and South Carolina until the age of 14, when the inadequacy of the education caused his father to remove him from school and tutor him at home. He had been introduced to Latin, Greek, and Hebrew and was well prepared by the time he entered college. Will wished to attend a large University, but his father thought his son at age 16 was too young and convinced him to attend Davidson College in North Carolina, a school for which he developed a lifelong affection. While in graduate school at Yale University, Rose decided on the branch of chemistry he would pursue, which was biochemistry, under the guidance of Lafayette Mendel in the Sheffield Scientific School. In 1911, upon completion of his Ph.D. thesis, Rose left Yale for an instructorship in physiological chemistry at the University of Pennsylvania, followed by advanced study with Franz Knoop at the University of Freiburg and then a faculty position at the University of Texas in Galveston before he became professor and head of the Division of Biochemistry in the Chemistry Department at the University of Illinois. This provided a permanent and very supportive home for his scientific career for the next 35 years. In research Rose displayed a gift for meticulous experimentation and for thoroughness and clarity in his publications. As a teacher he imbued students who attended his carefully prepared lectures with enthusiasm for biochemistry. The subject came alive with his engrossing stories about the early history of the field and the personalities involved. No mention of his remarkable ability as a teacher would be complete without reference to the seminars and lectures at which he imparted scientific knowledge and also entertained his audience as an incomparable raconteur. His research interests included the intermediary metabolism of amino acids, creatine, uric acid, and related compounds, and he was renowned for the discovery, isolation, and identification of a new amino acid as α-amino-β-hydroxy-n-butyric acid, which he named threonine (2McCoy R.H. Meyer C.E. Rose W.C. Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid..J. Biol. Chem. 1935; 112: 283-302Abstract Full Text PDF Google Scholar). This was the culmination of experiments in which rats failed to grow on diets containing the 19 previously known amino acids. Thus, painstaking efforts over many years led to the missing growth factor found in proteins and in hydrolysate fractions therefrom. When I arrived in Urbana to undertake graduate study in 1943, the identity of the 10 amino acids essential for growth in rats and the 8 essential for the maintenance of nitrogen equilibrium in the human (that is, male graduate students) was already known (3Rose W.C. Haines W.J. Johnson J.E. The role of the amino acids in human nutrition..J. Biol. Chem. 1942; 146: 683-684Abstract Full Text PDF Google Scholar, 4Rose W.C. The role of the amino acids in human nutrition..Proc. Am. Philos. Soc. 1947; 91: 112-116PubMed Google Scholar). It fell my lot to isolate, purify, and analyze amino acids and then feed them to fellow students enlisted as human guinea pigs in experiments involving daily nitrogen balance determinations. The diets consisted of the mixture of amino acids under study, the known vitamins, cornstarch, corn oil, sucrose, butter fat, inorganic salts, and Celluflour (a product providing roughage but no nutritive value, nitrogen, or flavor). The only taste thrill in this otherwise bland fare was a large brown “candy” containing a bitter liver extract as a possible source of unknown vitamins flavored with peppermint oil and sweetened with sugar. In those days the recruits were grateful for the free rations, the dollar a day they were paid, and the prospect of seeing their initials in print in Rose's widely read publications. The resulting papers established the quantitative requirements for the essential amino acids, the availability of some of the D-isomers orN-acetyl derivatives, and the role of cysteine and tyrosine in sparing methionine and phenylalanine, respectively. The morale of the subjects was maintained over many weeks by the prospect of collecting data for doctoral theses, the obvious importance of our findings for human welfare, and the infectious enthusiasm of Dr. Rose. An added benefit in my case was that, while consuming these daily rations, I had ample time to think about experiments on the metabolism of the essential amino acids I might pursue later in my career, as described below. Rose's students were somewhat in awe of the professor, perhaps wondering whether they could meet his exacting standards or hope to emulate the seeming ease with which he succeeded in all of his professional endeavors. They learned in time that behind his somewhat reserved and formal manner was a genuine warmth and an understanding that young scientists develop their full potential only by profiting from their mistakes. His research achievements earned him wide recognition and many honors. On the occasion of his 90th birthday his former students, colleagues, and friends assembled in Urbana to join him in the celebration. He was much surprised when presented with a handsome bronze plaque announcing the establishment of the William C. Rose Award and Lectureship. As indicated in Fig.1, the plaque to be given to all awardees shows his likeness and a sketch of the Noyes Laboratory with the structures of the essential amino acids and the stereochemistry and crystal structure of threonine, with a quotation and chart from his classical 1935 paper published in the Journal of Biological Chemistry (2McCoy R.H. Meyer C.E. Rose W.C. Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid..J. Biol. Chem. 1935; 112: 283-302Abstract Full Text PDF Google Scholar). This award, now administered by the American Society for Biochemistry and Molecular Biology, has been given annually, and the lectures are presented at the Society's national meetings. Until his death at age 98, Will Rose took a keen interest in those selected for the award named for him. He and his wife Zula exerted a wonderfully positive influence on all who knew them and took a personal interest in the 90 graduate students who studied under him, of whom 56 received the Ph.D. degree. In later years he often commented on his happy family life until his wife's death in 1965, his exciting professional life, and the thrill of watching his students grow into professional stature. In the first paper of this series on Reflections, Arthur Kornberg (5Kornberg A. Remembering our teachers..J. Biol. Chem. 2001; 276: 3-11Abstract Full Text Full Text PDF PubMed Google Scholar) has written perceptively of his deep admiration for Severo Ochoa, whom he knew particularly well from his stay in that in and their until death in I only on my to that exciting University the a years as a faculty at the University of I had not earned an but came to that a knowledge of was to progress in my studies on amino acid at the University of that the was the in at that and I on that and was most to have an research studying the of and and led to the and of now from Coon M.J. A. of acid and properties of Biol. Chem. Full Text PDF PubMed Google Scholar). 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