Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells.
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PMID:Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by alpha-chymotrypsin. 715 Feb 39

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of molecular weight 39 kDa/subunit having noncovalently bound NAD acting as cofactor. Denaturation by 8 M urea at pH 7.0 causes 85% loss of its secondary structure and dissociation of its constituent molecules. Dilution of the denaturant by buffer at pH 8.5 leads to functional reconstitution of the dimeric holoenzyme. The refolding process is biphasic: after 2 min an equilibrium conformer is formed having 72% of its native secondary structure and by 60 min reactivation becomes complete. The early intermediate has lower energy of activation against thermal denaturation than the reactivated state. Patterns of trypsin digestion suggests a native like structure of this intermediate. Variation of solvent viscosity and ionic strength and inclusion of proline cis-trans isomerase in the refolding process do not alter kinetics of reactivation. Moreover, unaltered kinetics of reactivation against variation of temperature, pH, and duration of denaturation strongly suggests absence of proline cis/trans isomerization. Measurement of kinetics of (i) recovery of tertiary structure by protein fluorescence; (ii) incorporation of NAD from quantitation of bound cofactor; (iii) formation of substrate binding site by specific interaction with extrinsic fluorophore 1-anilino-8-naphthalene sulfonic acid and quenching by 5'-UMP, a competitive inhibitor; and (iv) recovery of activity indicate that they are all comparable. It appears that internal rearrangement of the protein during refolding, shielded from solvent, is the rate-limiting step of generation of cofactor binding site which ultimately leads to maturation of the holoenzyme structure.
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PMID:UDP-galactose 4-epimerase from Escherichia coli: formation of catalytic site during reversible folding. 1143 50

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.
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PMID:UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site. 1552 57

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a homodimer containing one catalytic site and one NAD(+) as cofactor per subunit. One 5'-UMP, a competitive inhibitor, binds per dimer of epimerase as isolated and causes inactivation. Addition of 0.2 mm inhibitor to the enzyme in vitro leads to three sequential steps: first, the inhibitor binds to the unoccupied site; second, the inhibitor bound ex vivo is displaced allosterically; and finally, both sites are occupied by the inhibitor. These reactions have been monitored by kinetic lag in substrate conversion, coenzyme fluorescence, protection against trypsin digestion, and reductive inhibition. The transition profiles indicate the existence of a stable intermediate with one inhibitor-binding site remaining unoccupied. Reductive inhibition of this intermediate reduced the activity to 58% +/- 2%, with modification of one catalytic site. A change of conformation of the epimerase upon binding with substrate or inhibitor was evident from fluorescence emission spectra. The epimerase demonstrated a biphasic Michaelis-Menten dependency. The epimerase devoid of 5'-UMP showed a Michaelis-Menten dependency that can be explained by assuming simultaneous operation of two catalytic sites. A monomeric form of the epimerase was devoid of such regulation. The inhibitory profile of 5'-UMP also suggested negative cooperativity. Incubation of the epimerase with combinations of substrate analogs rendered one of the sites inactive, supporting the presence of two functional and regulated catalytic sites. Dissimilar kinetic patterns of the reconstituted enzyme after treatment with p-chloromercuribenzoate indicated stability of the dimeric enzyme against fast association-dissociation, which could otherwise generate multiple forms of the enzyme with functional heterogeneity.
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PMID:UDP-galactose 4-epimerase from Kluyveromyces fragilis--catalytic sites of the homodimeric enzyme are functional and regulated. 1984 83

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45 degrees C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.
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PMID:Presence of bound substrate in lactate dehydrogenase from carp liver. 2280 33