Biomedical subjects
Publications and source records attributed to E T Kaiser.
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The adenosine 3",5"-monophosphate (cAMP)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine heart is characterized. That the ATPase activity is intimately associated with the catalytic subunit of the enzyme is suggested by the following: (i) the similar dependences of ATPase and protein kinase activities on cAMP; (ii) the dissociation of ATPase activity from the holoenzyme on addition of cAMP and its co-elution with the catalytic subunit on gel filtration chromatography; (iii) the similarity of the relative effectiveness of divalent metal ions in ATPase and protein kinase catalysis; and (iv) the correspondence of kinetically determined Km(MgATP) and Ki(MgADP) values with thermodynamic dissociation constants determined by equilibrium dialysis. The hydrolysis of ATP is stimulated 10- to 20-fold by cAMP in the holoenzyme. The molar specific activity of the catalytic subunit ATPase is approximately 0.7 min-1 with Km(MgATP) = 5 muM. MgADP is a competitive inhibitor of the reaction with a Ki value of approximately muM. The order of the relative effectiveness of metal ions for both ATPase and peptide kinase activities is Mg2+ greater than Mn2+ greater than Ca2+. A possible interpretation of these observations is that the role that the metal ion plays is more directly manifested in bond-breaking than in bond-forming.
The reinvestigation of the affinity chromatographic method of purifying papain has been carried out. It has been reported that papain could be purified by taking advantage of the affinity of the enzyme for the insolubilized peptide inhibitor, agarose-Gly-Gly-Tyr(Bz)-Arg. Using pure tetrapeptide obtained commercially and standard coupling procedures, a significant purification of papain could not be achieved. Both active and nonactivatible enzyme bound to a column prepared in this manner were eluted together by the use of deionized water. An affinity medium with properties similar to those reported by Blumberg et al. was obtained by removal of the benzyl group on tyrosine prior to coupling with agarose. The deprotected tetrapeptide was also synthesized by an independent route and inhibition constants for the binding of the protected and deprotected tetrapeptide to papain were determined in kinetic experiments.
In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms. Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast. The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others. However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography. When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content. In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present. The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined. In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km. On the other hand, the deacylation rate constant, k3, is affected only slightly.
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The spectrophotometric titration of SH groups in adenosine 3',5'-monophosphate (cAMP) dependent protein kinase from bovine heart muscle with 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB) is described. The holoenzyme (R2C2) contains 16 SH groups, 12 of which react with DTNB in the native enzyme. The SH groups are distributed 2 per catalytic (C) and 4 per regulatory (R) subunit. The binding of cAMP to the holoenzyme or isolated R subunit prevents the reaction of one SH group per R subunit. Modification of SH groups, however, has only a small effect on cAMP binding to R. Reaction of the C subunit with DTNB results in less than 95% loss of catalytic activity. The kinetics of the DTNB reaction and the reversal of the inactivation process by treatment with dithiothreitol suggest that the inactivation is associated with SH group modification. Inactivation studies with the holoenzyme show that: (1) the R subunit inhibits DTNG inactivation of the C subunit in the absence of cAMP; (2) the rate of inactivation of the dephosphoholoenzyme in the presence of cAMP is considerably faster than that of the free catalytic subunit; and (3) the rate of inactivation of the phosphoholoenzyme in the presence of cAMP is faster than that of the C subunit but slower than the dephosphoholoenzyme. The results are interpreted as evidence for a significant interaction of the R and C subunits in the presence of saturating concentrations of cAMP. This interaction is modulated by the state of phosphorylation of R. To account for the inactivation data, a short-lived ternary complex containing R, C, and cAMP is postulated to be in rapid equilibrium with the subunits.
The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.
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The synthesis and characterization of protein proteinase inhibitor homologues with variations in the amino acid composition in the vicinity of the reactive site should aid the understanding of the mechanism by which inhibition of enzymatic activity occurs. A homologue inhibitor in which the reactive-site residue Ala-16 of basic pancreatic trypsin inhibitor (Kunitz) (BPTI) is replaced by Phe has been synthesized to study the effect of this replacement on the dissociation constants of the enzyme-inhibitor complexes. The replacement of Ala-16 by Phe causes a dramatic increase in the K1 value of the trypsin-BPTI complex while that of the chymotrypsin-BPTI complex remains essentially the same. This cannot be explained simply in terms of increased steric crowding. The Phe replacement probably causes a small change in the local conformation of the reactive site of the inhibitor which leads to a large decrease in the stability of the very tight trypsin-BPTI complex. This conformation change apparently can be tolerated in the less tightly bound chymotrypsin-BPTI complex. On the basis of the known structure of BPTI, a cyclic heptadecapeptide containing one disulfide bond was synthesized as a model inhibitor in order to determine if a smaller peptide can be designed to act as a highly efficient inhibitor for trypsin. This heptadecapeptide which contains all of the amino acid residues of BPTI taking part in the interaction of the proteinase inhibitor with trypsin binds 3 X 10(7) time more weakly to the enzyme than native BPTI does. It thus appears that even though only a small part of the inhibitor molecule enters directly into interaction with the enzyme, the remaining portions of the molecule which hold the structure of the inhibitor rigid are essential for the strong interaction.
By the use of sulfite ester substrates together with hydroxylamine as a highly reactive trapping agent, we have been able to obtain strong evidence for the intermediacy of enzyme-bound anhydride species in the pepsin-catalyzed hydrolysis of these substrates. From our observations that in the trapping experiments hydroxamate functions are introduced at the beta-carboxylates of Asp-32, Asp-215 and at least one additional Asp residue, it appears that several reactive carboxylate species can function as nucleophiles against sulfite esters, leading to the formation of anhydride species. Because the location of the hydroxamate incorporated into pepsin other than at the Asp-32 and Asp-215 residues is unknown, it remains conceivable that, at least for the action of pepsin on sulfite substrates, there are two distinct active site regions. If the possibility is considered that peptides possessing common amino-terminal residues but different acyl residues may bind productively in different fashions so that in some cases the beta-carboxylate of Asp-32 acts as the attacking nucleophile while in others the beta-carboxylate of Asp-215 acts in this way (as has been observed for sulfites), much of the confusion in the literature concerning the reactions of pepsin with peptidase may be explained.
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The use of polyethyleneimine-cellulose thin layer sheets to follow the phosphorylation of histone and decomposition of ATP catalyzed by an adenosine 3':5'-monophosphate (cyclic AMP)-stimulated protein kinase, protein kinase I, has made possible a more detailed analysis of the time course of these reactions than has been achieved previously be observing only recovered phosphorylated protein. When [gamma-32P] ATP was employed, significant error was introduced by the presence of 32Pi at the solvent front on these sheets, and this limited the accuracy of the available information. However, the analysis of assays performed with [U-14C] ATP was straightforward and appeared to have an accuracy comparable to that of the present standard assay. This appears to be the first use of [U-14C] ATP to assay protein kinases. Our physical characterization of protein kinase I showed it to be a homogeneous protein species by polyacrylamide gel electrophoresis, sodium dodecyl sulfate gel electrophoresis and analytical ultracentrifugation. Kinetic studies with protein kinase I indicated the absence of histone phosphatase and cyclic AMP phosphodiesterase activity. Furthermore, the ATPase activity seen is believed to be intimately associated with the protein kinase action, particularly in view of the observed dependence of the rate of Pi production on the presence of cyclic AMP. The kinetic data for the phosphorylation of histone catalyzed by protein kinase I under full stimulation by cyclic AMP are consistent with a double displacement mechanism.
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