More of Phosphorylase's Structure Known Enzyme fragment containing 14 amino acids goes through same interconversion that phosphorylase does More details are now available on the structure of phosphorylase, the enzyme that triggers the first step of the breakdown of stored tissue sugar (glycogen) into available glucose. Four biochemists at the University of Washington have added eight amino acids [Biochem., 3, 542 (1964)] to a known sequence of six amino acids within the phosphorylase molecule. This sequence of 14 amino acids, the Washington scientists find, makes up an active site. This site takes on or gives up phosphate (H 2 PO- 3 ) as phosphorylase assumes one of two characteristic forms: It becomes either more active or less active in catalyzing glycogen breakdown. These 14 amino acids are a large enough fraction of the massive (molecular weight 125,000) phosphorylase subunit to account for the change between the two forms, say Dr. Chris Nolan (now at Abbott Laboratories), Dr. William B. Novoa (now at the University of Kansas), Dr. Edwin Krebs, and Dr. Edmond Fischer. Phosphorylase regulates one step in the physiology of glucose. Glucose's metabolic pathway begins with conversion of dietary glucose to glucose 6-phosphate (glucose-6-P). Glucose6-P can undergo glycolysis and become an energy source. Or it can be converted via glucose-1-P to glycogen. As glycogen, it is available for conversion—back through glucose-1-P, glucose-6-P, and other steps—in the energy liberation process. Phosphorylase is active during the first step of glycogen breakdown—conversion to glucose-1-P. The enzyme itself can be in the form of phosphorylase a or phosphorylase b. Phosphorylase a is a much more active catalyst than is phosphorylase b. Whether phosphorylase exists as a or b depends, in turn, on two other enzymes: phosphorylase b kinase and phosphorylase phosphatase. The kinase favors the reaction of phosphorylase b with adenosine triphosphate (ATP) to form phosphorylase a and adenosine diphosphate ( A D P ) . The phosphatase favors breakdown to phosphorylase b and phosphoric acid. This transfer intrigues biochemists. Not only is it important in metabolic
regulation, but only a few known enzymes exist in two forms, the Washington workers say. Digestive enzymes are first produced in inactive forms (called zymogens), converted to active agents, and then digested. But the zymogens can't be reconstructed from the active form. Thus, phosphorylase is unique. Properties. The gross properties of phosphorylase are known. Its smallest subunit contains 1025 amino acids and has a molecular weight of 125,000. Phosphorylase b is made up of two of these units. Phosphorylase a has four of these units plus four moles of inorganic phosphate (H 2 PO~ 3 ). Dr. Fischer, Dr. Krebs, and their co-workers began studying the b to a reaction in the 1950's. Since the subunit of 1025 amino acids is so massive,
total characterization of the protein was out of the question. Instead, the Washington workers began looking for the nature of the site phosphorylated when the inactive b is converted to a. By 1959 they had determined that the phosphate is attached to the amino acid serine; this serine is in a sequence of six amino acids: lysine-glutamine-isoleucine-serine phosphate-valine-arginine. The Washington biochemists split this sequence of six amino acids from phosphorylase by a series of biochemical steps. First they formed labeled phosphorylase a from phosphorylase b and (P 3 2 ) ATP. Then they allowed the proteolytic enzyme trypsin to chop up the radioactive phosphorylase a and collected the radioactive residues. Early tests of the phosphorylated six-acid fragment showed that the phosphate was slowly split off from the fragment by phosphorylase phosphatase, much like the a to b transfer. However, this fragment was not rephosphorylated by phosphorylase b kinase. Thus the six-acid fragment
14-Amino Acid Fragment Undergoes Phosphorylase a to b Interconversion fhcripkruJaAe b KtwaAe
W + 4 ATP Tko4|>kor\daAe b
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The 14-amino acid fragment (containing H2 P0 3 ~ ) of phosphorylase undergoes the same a to b interconversion depicted above that phosphorylase itself does
Phosphorylase Regulates One Step in Physiology of Glucose KCvutde
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was somehow not representative of the active site. The four Washington scientists then sought to chop a bigger part out of the phophorylase unit and test it with the kinase and phosphatase. They did this in much the same way as before. First, they formed labeled phosphorylase a from phosphorylase b and radioactive (P 3 2 ) ATP; then they split off fragments with proteolytic enzymes. The proteolytic enzymes are specific, although the size of the fragment they split off depends on the protein being broken up and the various reaction conditions. Dr. Fischer and his co-workers tried several enzymes before finding one—chymotrypsin—that produced the larger fragment. With this enzyme, they were able to isolate the 14-amino acid fragment. The structure of this fragment is serine-aspartic acid-glutamine-glutamic acidlysine - arginine - lysine - glutamineisoleucine-serine phosphate-valine-arginine-glycine-leucine. This tetradecapeptide will undergo the b to a and a to b transformations. Phosphorylase phosphatase will dephosphorylate it at about 2 to 3 % of the rate of dephosphorylation of phosphorylase a. Similarly, phosphorylase b kinase will rephosphorylate the peptide slowly in the presence of (P 3 2 ) ATP. Chymotrypsin. The entire protein is broken down by chymotrypsin. An important simultaneous finding, the Washington workers feel, is that the rate of chymotrypsin's release of the (P 3 2 ) -labeled portions of the protein is greater than the over-all rate of protein breakdown. This suggests that the site that is phosphorylated is particularly exposed, or less highly organized than is the rest of the molecule. The Washington workers are quick to point out that they have only touched on the question of the chemical nature of phosphorylase. Why the enzyme grows from two to four units on phosphorylation is still another question. The acids around serine are positively charged, and H 2 PO _ 3 tends to neutralize this charge, possibly contributing to a four-from-two transfer. But this doesn't fully account for the situation. In fact, why b is in two units remains unknown. The structure of the 14-unit fragment and its definition as an exposed part of the molecule give no information on why phosphorylase a is a better glycogen breakdown agent than is b.
