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Reaction of Dopa decarboxylase with alfa-methylDopa leads to an oxidative deamination producing 3,4-dihydroxyphenylacetone, an active-site directed affinity label
No full textDopa decarboxylase (DDC) catalyzes the cleavage of alpha-methylDopa into 3,4-dihydroxyphenylacetone and ammonia, via the intermediate alpha-methyldopamine, which does not accumulate during catalysis. The ketone has been identified by high-performance liquid chromatography and mass spectroscopic analysis, and ammonia by means of glutamate dehydrogenase. Molecular oxygen is consumed during the reaction in a 1:2 molar ratio with respect to the products. The kcat and Km of this reaction were determined to be 5.68 min-1 and 45 microM, respectively. When the reaction is carried out under anaerobic conditions, alpha-methyldopamine is formed in a time-dependent manner and neither ammonia nor ketone is produced to a significant extent. The reaction is accompanied by a time- and concentration-dependent inactivation of the enzyme with kinact of 0. 012 min-1 and Ki of 39.3 microM. Free 3,4-dihydroxyphenylacetone binds to the active site of DDC and inactivates the enzyme in a time- and concentration-dependent manner with a kinact/Ki value similar to that of alpha-methylDopa. d-Dopa, a competitive inhibitor of DDC, protects the enzyme against inactivation. Taken together, these findings indicate the active site directed nature of the interaction of DDC with 3,4-dihydroxyphenylacetone and provide evidence that the ketone generated by the reaction of DDC with alpha-methylDopa dissociates from the active site before it inactivates the enzyme. Inactivation of the enzyme by ketone followed by NaB3H4 reduction and chymotryptic digestion revealed that the lysine residue which binds pyridoxal 5'-phosphate (PLP) in the native enzyme is the site of covalent modification. Together with the characterization of the adduct released from the inactivated DDC, these data suggest that the enzyme is inactivated by trapping the coenzyme in a ternary adduct with ketone and the active site lysine. As recently reported for serotonin (5-HT) [Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem. 271, 23954-23959], the conversion of dopamine (DA) into 3,4-dihydroxyphenylacetaldehyde and ammonia catalyzed by DDC is accompanied by irreversible loss of decarboxylase activity. However, the comparison between the absorbance, fluorescence, and CD features of DDC after 5-HT- or 3, 4-dihydroxyphenylacetone-induced inactivation shows that a different covalent adduct is formed between either of these two molecules and DDC-bound PLP
Affinity labelling of pig kidney Dopa decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active site cysteine
No full textDifferent susceptibility of the two dimers of ribonuclease A to subtilisin. Implications for their structure.
No full textRNase A and its minor and major dimers were digested with subtilisin under controlled conditions. The major dimer was found to be slightly more resistant, the minor dimer markedly less resistant to subtilisin than monomeric RNase A. Two S-proteins formed for each RNase A species, one starting with Ser-21 the other with Ser-22. Their relative proportions indicate that the structure of the minor dimer, whose identity with that of a RNase A dimer shown to be 3D domain-swapped is strongly suggested by recent work [S. Sorrentino et al. (2000) FEES Lett. 466, 35-39], makes its peptide bond between Ser-21 and Ser-22 more accessible to subtilisin than it is in RNase A and its major dimer. Moreover, (i) both subunits constituting the minor dimer are more susceptible to subtilisin than monomeric RNase A, and (ii) the susceptible bonds in one of its two exchanging N-terminal arms are more accessible to the protease than in the other. The properties of the major dimer suggest that its structure could be different. (C) 2001 Elsevier Science B.V. All rights reserved
A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III RID A-2020-2010 RID A-4573-2009
No full textThe design of chimeric proteins is a major held of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors. (C) 2000 Academic Press
Is pantetheinase the actual identity of mouse and human vanin-1 proteins?
No full textPantetheinase is an amidohydrolase involved in the dissimilative pathway of CoA, allowing the turnover of the pantothenate moiety. We have determined the N-terminal sequence as well as the sequences of a number of tryptic and chymotryptic peptides of the protein isolated from pig kidney. These sequence stretches were used as probes to search in the SwissProt database and significant similarities were found with a GPI-anchored protein (mouse vanin-1, with a suggested role in lymphocyte migration), with two putative proteins encoded by human cDNAs (VNN1 and VNN2) and with human biotinidase. On the basis of sequence similarity, we propose that vanin-1 and VNN1 should be identified as pantetheinase
A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III. Biochem Biophys Res Commun. 2000 Sep 7;275(3):817-20. PubMed PMID: 10973804
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Dopa decarboxylase: the pig kidney enzyme primary structure and relationship to other amino acid decarboxylases
No full textMicro-dominii di inibizione enzimatica in grado di proteggere proteine espresse in sistemi eterologhi dalla degradazione proteolitica
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