RESEARCH
1
the Medical Biological Laboratory of the National Defense Research Council, Rijswijk Z.H., the Netherlands, note that phosphorylation studies "have established the significance of the serine residue at the active site," and that in chymotrypsin and other esterases "the serine hydroxyl functions as acyl and phosphoryl acceptor" [/. Cellular Comp. Physiol, 54, Suppl. 1, 231 (1959)]. The Dutch workers define "active site" as that region "of the enzyme surface where the substrate is localized and activated during the enzymic action."
Bifunctional Substrate Aids Enzymology Method confirms importance of unhindered methionine for normal chymotrypsin activity A method by which enzymes may be selectively modified has been designed by Dr. W. B. Lawson and Dr. H. J. Schramm of the Division of Laboratories and Research, New York Department of Health, Albany. Application of the technique to the hydrolytic and proteolytic pancreatic enzyme chymotrypsin verifies the importance of unhindered methionine in this enzyme for its proper activity. The technique Dr. Lawson and Dr. Schramm developed [JACS, 84, 2017, (1962)] uses a bifunctional reagent that attaches to an amino acid side chain near the enzyme's active site. Thus they find that when the p-nitrophenyl ester of bifunctional bromoacetyl-a-aminoisobutyric acid is used as the substrate for chymotrypsin, esterase activity drops about 80% (using a 10-fold excess of the ester at pH 5). Reincubation of the inhibited chymotrypsin with additional p-nitrophenyl bromoacetyl-oj-aminoisobutyrate doesn't reduce its activity further, the New York scientists note.
The work of the New York group is part of a continuing study being carried out in the U.S. and abroad to explain the nature and mechanism of enzymes. For example: • Dr. A. K. Balls, Dr. M.-D. F. Nutting, and E. F. Jansen of the U.S. Department of Agriculture, Albany, Calif., showed esterase and proteinase activity of chymotrypsin to be completely inhibited by diisopropyl fluorophosphate ( D F P ) , the phosphorus becoming firmly bound to the enzyme in a mole-to-mole ratio [/. Biol. Chem., 179, 189,201 (1949)].
Complex. Dr. Lawson and Dr. Schramm show that chymotrypsin is first acylated at the active site (the serine residue) with liberation of p-nitrophenol. The second functional group of the substrate then links with a neighboring methionine residue. The resulting complex is stable to dialysis, pH change, and lyophilization. Alkylation of the methionine may occur while the bromoacetyl-oj-aminobutyric acid is still covalently bound to the serine residue or shortly after the normal hydrolysis of the acylserine bond, the New York scientists say. Dr. Lawson and Dr. Schramm prove that the serine residue of the low activity chymotrypsin complex is free and capable of functioning as an enzyme active site by treating the
• D r . N. K. Schaffer, Dr. W. H. Summerson, and S. C. May, Jr., during their studies at the Enzyme Chemistry Branch of the Chemical Corps Medical Laboratories, Army Chemical Center, Md., found that DFP's phosphorus becomes bound to chymotrypsin's serine residue [J. Biol. Chem., 202, 67 (1953)]. • Dr. J. A. Cohen and co-workers at
Methionine Residue Reacts With Bifunctional Substrate 0 / C-O-CHo-C-H
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When chymotrypsin is exposed to a bifunctional ester substrate, the ester is first cleaved to form the acylated enzyme at the active (serine) site. Interaction then takes place between the bifunctional acyl radical and a neighboring methionine residue either before or shortly after breaking of the acyl-serine bond. The resulting stable complex has only 2 0 % of the original enzyme activity. How the acylated methionine residue reduces the over-all activity of the enzyme isn't known. It might be due to steric hindrance or to a redistribution of the charge on the protein 38
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JUNE
25,
1962
H0-CH2-C^H
CHYMOTRYPSIN
/ / ** r CH3 H I OOC-C-N-C-CH,-^
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complex with diisopropyl fluorophosphate. A 1:1 molar binding of phosphorus results, accompanied by a drop in activity to less than 1%. Amino acid analysis of chymotrypsin and of the 20% active chymotrypsin complex shows that the only difference between the two is a decrease in methionine content from the normal value of 2 for chymotrypsin to 1 in the complex. In addition, hydrolysis of the complex gives a-aminoisobutyric acid in a mole-to-mole ratio, Dr. Lawson and Dr. Schramm point out. Mechanism. The exact mechanism by which the acylated methionine reduces chymotrypsin activity is not known, Dr. Lawson and Dr. Schramm say. They suggest that the bulky acyl group on the methionine may hinder ester or amide substrates from approaching the serine active site. The drop in activity might also result from a change in the electric charge distribution on the protein, they note. The importance of the correct charge distribution on the enzyme protein has been recognized for some time. For example, Dr. Leon W. Cunningham of Vanderbilt University bases his proposed mechanism of the action of hydrolytic enzymes on hydrogen bonding and charge transfers between the substrate's carbonyl group and the protein's serine and histidine (imidazole) fractions [Science, 125, 1145 (1957)]. The New York scientists, however, believe it unlikely that the sulfide group of the critical methionine behaves as a neighboring nucleophile at the active site of chymotrypsin because this activity should disappear on alkylation. Dr. Lawson and Dr. Schramm point out that their results confirm findings of others. For example, Dr. D. E. Koshland, Jr., Dr. W. J. Ray, Jr. (now at Purdue University), M. Katsoulis, and H. G. Latham, Jr., of Brookhaven National Laboratory and the Rockefeller Institute show that methionine as well as histidine is involved in the catalytic activity of chymotrypsin [JACS, 82, 4743 ( I 9 6 0 ) ] . These workers, using an "all-or-none" assay, have also shown that an enzyme molecule having one of its methionine residues modified has only a fraction of the activity of the native enzyme. And Brookhaven's Dr. Koshland and Dr. D. Strumeyer say that chymotrypsin in which one methionine has been oxidized with hydrogen peroxide shows 30% of the activity of the native enzyme.
Superconductor Reverses Magnetic Field Change in magnetic field's sign, predicted in 1953, supports theory of superconductivity Reversal of a magnetic field's direction as it passes through a superconducting thin film has been observed at International Business Machine's research laboratory in Zurich, Switzerland. Observation of the sign reversal by IBM's M. K. E. Drangeid and Dr. R. Sommerhalder [Phys. Rev. Letters, 8, 467 (1962)] provides direct physical evidence that some unexplored predictions of the theory of superconductivity developed by the University of Illinois' Dr. John Bardeen and co-workers are valid. Sign reversal was first predicted by Dr. A. Pippard of Cambridge University in 1953. The observation by the IBM scientists was made during an experiment designed to show how magnetic fields penetrate superconducting thin films. They made a thin-film metal cylinder having a wall 0.007 inch thick by evaporating the metal onto polished borosilicate glass substrates precoated with S n 0 2 at a pressure of 10 - 9 torr; deposition rate is several hundred A. per second. Annealing temperatures do not exceed room temperature. Diameter of the cylinder is 0.8 inch, and it's 6 inches long. A coil of wire inside the cylinder serves as a sensing (or pickup) device. A second wire coil goes around the outside of the cylinder.
An a.c.-generated magnetic field (at a frequency of 1.1105 cycles per second) is applied parallel to the axis of the cylinder. Current for the exciting field is produced by a sine wave generator. The external magnetic field penetrates the cylinder wall and sets up a small field inside the cylinder. The field inside the cylinder (tuned with an external capacity and amplified in a tuned amplifier) is measured by the internal sensing coil and compared with the known external field. The comparison is made by displaying signals from both the external and internal fields on a dual-beam cathode ray oscilloscope. Both the amplitudes and the phases of the two voltages are compared. The oscillograph photos show the exciting current and the corresponding pickup voltage, taken at 3.41° K. and 2.88° K., respectively. Taking the current signals as reference, the 180° shift in the corresponding voltage signals is immediately evident, the IBM scientists say.
SIGN REVERSAL. These two oscillograph photos show reversal of the magnetic field in a superconducting thin film. The heavy line in both photos is the magnetic field as it's picked up (at 2.88° K.) by a sensing coil; the thin line is the reference signal, at 3.41° K. At the top is the magnetic field before reversal; on the bottom, the magnetic field is reversed. The observation, made at IBM's labs in Zurich, Switzerland, adds support to the theory of superconductivity. Sign reversal was first predicted in 1953. The experiment was done with a thin-film metal cylinder having a wall 0.007 inch thick, 0.8 inch in diameter, and 6 inches long. A coil inside the cylinder serves as the sensing device; a second wire coil goes around the outside of the cylinder. The comparison is made by displaying signals from both the internal and external fields on the dual-beam oscilloscope. With the current signals as reference, the 180° shift Is evident JUNE
2 5,
1962
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