ACS Award Eli Lilly and Company Award in Biological
More light on Protein Structure Chemistry
PAUL aeon W h e n Paul Berg received his B.S. from Pennsylvania State University he was set to make his career in physio logical chemistry. But when he started his graduait* lutining at Western Reserve Uni versity, Berg's i η t e r e s t s shifted to the applications of enzymology in studies of in termediary metahol ism. The decision was evidently a wise one, for this year, at 32, Berg receives the Eli Lilly and Company Award in Bio logical Chemistry. Basis for •fi the ll en/.\matic studies on the mechanism of phosphorylation of nucleoside diphosphates, and his dis covery that adenyl aeylates are the intermediates in acti vating amino and fatty acids and in the synthesis of amino acid-ribonucleic acid compounds. Berg caught the eye of his professors at Western Reserve when, for his P h . D . thesis, he quite independently de veloped a soluble enzyme system for methionine synthesis. It contained many original observations on methyl group formation a n d served as a start i n g point for subsequent studies carried o u t by others. Ilarland Wood, direc tor of the biochemistry department, Arnold Welch, former chairman of t h e pharmacology department, and others a t Western Reserve who watched Berg closely during these years think he was a n d remains one of the best graduate students in their memory. Instead of exploiting the start h e h a d m a d e in the intermediary metabolism of one-carbon compounds. Berg chose t o continue his training in the tech niques of enzyme chemistry as applied t o the metabolism of nucleotides. lie spent his first fellowship year ( 1 9 5 2 - 5 3 ) in Herman Kalckar's labo ratory in Copenhagen, where h e dis covered a new and unexpected reaction of considerable significance—the nu cleoside diphosphate kinase reaction. T h e transfer of t h e terminal phosphate of a nucleoside triphosphate to a nucleo side diphosphate has now been found in all cells and a t a high level of ac
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Clues come from r o t a t o r y dispersion, critical size of helixes; side groups or protein chain affect rotation
"S 5 ? - Κ ACS NATIONAL MEETING Physical C'tonistry
tivity. Knowledge of this reaction has required revision of many accepted notions of the specificity of adenosine triphosphate ( ATP ) and is fundamental to any interpretation of transfer reac tions involving di- and triphosphates. In 1953, Berg moved to Washington University's school of medicine, St. Louis, Mo., where he studied under Arthur R o m b e r g . One year later he was appointed a Cancer Research Scholar, and chose to study the mecha nism of the inorganic pyrophosphateATP exchange in the acetate-CoA kinase reaction. He soon succeeded in purifying the responsible enzyme and demonstrating an acetate-dependent exchange. These studies led to the con cept of the acyl adenylate intermediate. More recently, Berg has achieved the first well characterized and quanti tative syiuliesis iif ai"! amino acyl RNA compound. This important metabolic intermediate was prepared using highly purified amino acid-activating enzymes. His work has moved rapidly and de cisively toward the characterization of the amino acid-polynucleotide linkages and the specificity of the reactants. Washington University appointed Berg assistant professor in 1955 and associate professor in 1957. In June he will become associate professor of biochemistry at Stanford University.
JLVKSEARCH into biologically important molecules gets more interesting as more data appear on the shape of protein chains. Optical rotation of polarized light by a simple amino acid ( polypep tide) gives preliminary data on min imum chain length needed by a protein helix to be stable—another clue to pro tein structure. "Whether the helical chain configura tions of amino acids in proteins are right or left handed needs to b e known before a complete three dimensional structure of an enzyme site can b e de scribed. Analysis of optical rotation measurements may lead to a n answer on the particular sense (twist) of pro tein helixes, Ignacio Tinoco, Jr., of the University of California told the Divi sion of Physical Chemistry. Usually, measurements of optical ro tation are m a d e on molecules in solu tion. Since the molecules are rotating randomly, results are average values for rotation along different directions. T h e approach has been to orient the molecules in an electric field and meas ure separately optical rotation along the helical axis and perpendicular to the helical axis, says Tinoco. Side groups on the protein chain significantly influence the optical ro tation, according to Tinoco. Experi mental work with poly-benzyl-L-glutamate oriented in an electric field shows: • Optical rotation along the helical axis is large and positive. • Optical rotation perpendicular to the helical axis is large and negative. • Average rotation is small and its sign depends on the wave length of the polarized light. On the basis of calculated rotation for simple polypeptides, agreement with experimental results for complex poly peptides appears qualitative. But cal culations for complex proteins with
RESEARCH large and complicated side chains (similar to benzyl glutamate), are al most hopeless, says Tiuoco. Thus cal culations of optical rotation cannot be used yet to assign the sense of the helix because of the influence of side chain groups. This problem might be over come, Tinoco suggests, through more careful analysis of how the optical rotation depends on the wave length of light ^ Helixes A p p e a r Short. T h e helical chain configuration in proteins is sta bilized by hydrogen bonds between C—Ο and Ν—Η groups in the mole cules. But length of helical regions in some protein molecules must b e limited by cross-linking and other steric effects. So the question arises as to how short a helix can b e and still be stable. For a specific model protein, poly glutamic acid ( P G A ) , Jon Applequist, now at the University of California, and Paul Doty at Harvard came up with a criti cal size of six amino acid units. Applequist and Doty partially race mized PGA and measured the amount of optical rotation, which in turn gives a measure of the amount of either the right or left hand type helix present. T h e technique is simple; first, poly-Lglutamic acid is heated at a high pH. T h e product contains random sequences of D and L amino acid units along the polymer chain. At low p H , PGA is helical, but the D and L· units each tend to form helixes of opposite handedness. Racemized PGA containing both types of units is helical only where a single type occurs in a sequence long enough to form a helix of the handedness pre ferred by that type, according to Apple quist. Thus the optical rotation of partially racemized PGA depends on the amount of helix of either handedness present. Therefore, says Applequist, rotation measures t h e fraction of amino acid units which are in sequences greater t-u *-u~ —i*-i^^l t i i c i i i LIIC; L11 v i t a l
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