Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Optimal conditions have been determined for the coupling of rat liver phenylalanine hydroxylase (PheH) to activated CH-Sepharose-4B. When 12 mg of ligand was reacted with 100 mg of matrix, 20% of the initial enzyme activity was covalently bound along with 55% of the protein. The coupled enzyme showed greater thermal stability from 50 degrees to 60 degrees after heating for 15 min, a lower optimum pH, 5.8, slightly less inhibition by Ag+, Cu+2, and Hg+2, and greater resistance to hydrolysis by alpha-chymotrypsin and protease. The uncoupled enzyme, however, exhibited greater storage stability than the covalently linked enzyme at 25 degrees after 24 hrs and at 0 degrees after 21 days. Alteration of the microenvironment by the introduction of sulfhydryl groups and positive and negative charged carriers during coupling of the enzyme either had no effect or markedly reduced hydroxylase activity.
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PMID:III. Covalent coupling of rat liver phenylalanine hydroxylase. 2 12

Activation of rat liver phenylalanine hydroxylase by limited proteolysis catalyzed by chymotrypsin was investigated with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure gel filtration. Both activation and proteolysis were decreased by the addition of the natural cofactor, (6R)-tetrahydrobiopterin. From chymotryptic digests of the hydroxylase carried out in the presence and absence of (6R)-tetrahydrobiopterin, several different enzyme species were isolated by high pressure gel filtration. One species (subunit Mr = 47,000) with unchanged hydroxylase activity was isolated from the chymotryptic digest in the presence of (6R)-tetrahydrobiopterin; it was derived from the native enzyme (Mr = 52,000) by cleavage of the COOH-terminal Mr = 5,000 portion of the native enzyme. In the absence of (6R)-tetrahydrobiopterin, another species (subunit Mr = 36,000) was isolated. In addition to modification at the COOH-terminal end of the molecule, this species also had lost a Mr = 11,000 fragment from the NH2-terminal end of the hydroxylase. The Mr = 11,000 fragment was shown to include the phosphorylation site of the enzyme. This Mr = 36,000 species was 30-fold more active than the native phenylalanine hydroxylase when assayed in the presence of tetrahydrobiopterin. These results suggest that the regulatory domain that inhibits hydroxylase activity in the basal state may be located at the NH2 terminus of the phenylalanine hydroxylase subunit.
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PMID:Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase. 394 27

Pure phenylalanine hydroxylase from rat liver can be activated by limited proteolysis with alpha-chymotrypsin. As with most other types of activation of this enzyme, including activation by exposure to lysolecithin, the increase in activity is expressed in the presence of the naturally occurring pterin cofactor, tetrahydrobiopterin, but not in the presence of synthetic pterin cofactors such as 6-methyltetrahydropterin. With the chymotrypsin-activated enzyme, we have demonstrated directly, using circular dichroism measurements, that the activated enzyme differs in conformation from the native enzyme. In addition to chymotrypsin, trypsin and a mixture of rat liver lysosomal proteases can also activate phenylalanine hydroxylase. The latter finding raises the possibility that activation of the enzyme by limited proteolysis may be a physiologically important process. In experiments carried out with phenylalanine in which all five hydrogens on the aromatic ring have been replaced with deuterium, and in the presence of tetrahydrobiopterin, we have been unable to detect a kinetic isotope effect with either the native hydroxylase or with the hydroxylase activated by limited proteolysis, or by exposure to lysolecithin. By contrast, with both native and activated enzymes, a small isotope effect was detected when 6-methyltetrahydropterin was used as the pterin cofactor.
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PMID:The activation of rat liver phenylalanine hydroxylase by limited proteolysis, lysolecithin, and tocopherol phosphate. Changes in conformation and catalytic properties. 650 8

The effects of phenylalanine and tetrahydrobiopterin on the limited proteolysis of rat liver phenylalanine hydroxylase by chymotrypsin have been examined. The presence of tetrahydrobiopterin inhibits the proteolytic activation of native phenylalanine hydroxylase. In contrast, phenylalanine causes a stimulation of proteolytic activation under these conditions. Neither phenylalanine nor tetrahydrobiopterin affect the rate of hydrolysis of a synthetic substrate by chymotrypsin. Both tetrahydrobiopterin and phenylalanine inhibit the release of soluble radioactivity from [32P]phosphorylated phenylalanine hydroxylase. These results confirm the existence of multiple conformational states of phenylalanine hydroxylase.
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PMID:Ligand effects on the limited proteolysis of phenylalanine hydroxylase: evidence for multiple conformational states. 683 60

Rat liver phenylalanine hydroxylase can be markedly activated by a variety of different procedures including those that lead to covalent modification of the enzyme, such as limited proteolysis by alpha-chymotrypsin and alkylation of sulfhydryl groups by N-ethylmaleimide, and those that lead to reversible changes, such as the interaction of the enzyme with lysolecithin and related compounds. These treatments not only lead to increased activity toward the normal substrate for the enzyme, phenylalanine, but they lead to even greater increases in activity toward normally poor substrates such as m-tyrosine. Activations of this type, therefore, in effect, appear to broaden the amino acid specificity of the enzyme. We have now found that the extent of the change in substrate specificity on activation of the enzyme is much greater than had been heretofore realized. Indeed, the lysolecithin-activated enzyme is able to act on nonaromatic amino acids such as methionine and norleucine, catalyzing their conversion to methionine sulfoxide and epsilon-hydroxynorleucine, respectively. These amino acids are also substrates for phenylalanine hydroxylase that has been activated by limited proteolysis with alpha-chymotrypsin and by reaction with N-ethylmaleimide. By contrast, the unactivated hydroxylase as little or no activity toward methionine and norleucine. Rat liver phenylalanine hydroxylase can also be activated by its substrate, phenylalanine. We have now found that the novel substrates, methionine and norleucine, are also able to activate the enzyme. On a molar basis, however, they are not as active as phenylalanine. A model that can account for the broadened substrate specificity of the activated enzyme is discussed.
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PMID:Specificity of amino acids as activators and substrates for phenylalanine hydroxylase. 717 61