Keep Nibbling at the Edges (2009)

A lifelong passion for natural history and an aptitude for chemistry had led me to study for a B.Sc. in biochemistry at University College London (UCL) from 1963 to 1966. It was during this period that the allosteric theory of enzyme regulation was put forward by Jacques Monod, Jean-Pierre Changeux, and Francois Jacob at the Institute Pasteur. This explained how the end products of a biosynthetic pathway exerted feedback control on the enzyme catalyzing the initial and rate-limiting step. Reading these papers sparked an interest in enzyme regulation and led me to become a graduate student in the UCL laboratory of Michael Rosemeyer, with whom I had carried out a short undergraduate project the previous year. Michael had offered a project to study the control of glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate shunt. I purified Glu-6-P dehydrogenase and determined its subunit composition (1.Cohen P. Rosemeyer M.A. Eur. J. Biochem. 1969; 8: 1-7Crossref PubMed Scopus (74) Google Scholar, 2.Cohen P. Rosemeyer M.A. Eur. J. Biochem. 1969; 8: 8-15Crossref PubMed Scopus (98) Google Scholar) because the “quaternary structure” of an enzyme was thought to be key to how it was regulated by allosteric effectors. The project gave me an excellent grounding in protein chemistry, but Glu-6-P dehydrogenase seemed merely to be regulated by the NADPH/NADP ratio, and it was clear that I needed to find a more challenging problem to work on. The idea of applying to Edmond (Eddy) Fischer for a postdoctoral position arose during a discussion with Prakash Datta, a professor in the department at UCL, and Bill Whelan, the Chairman of Biochemistry at the University of Miami. Bill had just founded FEBS Letters and had come to visit because Prakash had become the first Managing Editor of the journal. I had heard Eddy Fischer give several lectures at a protein chemistry summer school in Venice in 1967, and so Bill, who was a friend of Eddy, agreed to write a letter of support to accompany my application. However, my initial approaches were unsuccessful, and only after I had obtained a UK Science Research Council (subsequently renamed the Biotechnology and Biological Sciences Research Council) postdoctoral fellowship funded by NATO did Eddy agree to let me come. To this day, I therefore always re-evaluate very carefully persistent postdoctoral applicants who will not take no for an answer! In 1955, Eddy Fischer and Edwin (Ed) Krebs had discovered that glycogen phosphorylase was activated by a phosphorylation mechanism and inactivated by dephosphorylation. This was the first example of enzyme regulation by reversible phosphorylation, a finding that was to gain them a Nobel Prize 37 years later (Fig. 1). However, in October 1969, when my wife, Tricia, and I arrived in Seattle (Tricia to carry out postdoctoral research with Arno Motulsky in the Department of Genetics), protein phosphorylation was still considered to be a specialized control mechanism, largely confined to the regulation of glycogen metabolism. Three enzymes involved in this process were known to be regulated by phosphorylation, namely glycogen phosphorylase itself; its activator, phosphorylase kinase; and glycogen synthase. cAMP-dependent protein kinase (PKA) had been identified in 1968 in Ed Krebs' laboratory and was known to activate phosphorylase kinase and to inhibit glycogen synthase, explaining how adrenalin mobilized glycogen and suppressed its synthesis. The 2 years working in Eddy Fischer's laboratory shaped the rest of my career and was a fantastic experience. I was interested in how control by reversible phosphorylation had evolved and embarked on a study of the regulation of glycogen phosphorylase in a primitive vertebrate (the Pacific dogfish). However, the dogfish enzyme turned out to be disappointingly similar to its mammalian counterpart (in retrospect, not that surprising), and I even found that dogfish phosphorylase kinase could activate mammalian phosphorylase and that mammalian phosphorylase kinase could activate dogfish phosphorylase (3.Cohen P. Duewer T. Fischer E.H. Biochemistry. 1971; 10: 2683-2694Crossref PubMed Scopus (137) Google Scholar). It was these experiments that started to kindle my interest in phosphorylase kinase. Eddy's favorite project at the time was a study of the “glycogen particles” that could be isolated from rabbit muscle with the enzymes involved in glycogen metabolism and their regulation still attached. When glycogen particles were incubated with MgATP, there was a rapid “flash” activation of glycogen phosphorylase, provided that micromolar concentrations of calcium ions were included in the incubations. This was followed by an almost equally rapid dephosphorylation and inactivation once the ATP had been rapidly hydrolyzed by elements of the sarcoplasmic reticulum ATPase that contaminated the preparations. The requirement for calcium was explained when Ludwig Heilmeyer and Richard Haschke, the postdoctoral researchers working on this problem, showed that phosphorylase kinase was reversibly activated by calcium ions. Similar findings were made by Ed Krebs, who had left Seattle in 1968 to become the founding Chairman of the Department of Biological Chemistry at the University of California at Davis. (Ed returned to Seattle in 1978 to become Chairman of Pharmacology.) Another long-standing interest of the Fischer laboratory was the characterization of phosphorylase phosphatase and how it was regulated, but the isolation of this enzyme had proved a hard nut to crack partly because of its much lower low level of expression. Eddy was fond of bringing visitors into the lab and, after introducing them to Danielle Gratecos, the postdoctoral researcher then working on the phosphatase problem, would reveal his frustration at the lack of progress by saying, “She (Danielle) purifies and purifies, and after the last step, the phosphatase activity comes out in a region of the column where there is no OD (absorbance at 280 nm); you know, it might not even be a protein!” (The phosphorylase phosphatase activity was also very resistant to tryptic digestion.) Somehow, I felt the phosphatase problem could not be as intractable as all that. 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