Evidence That the Nature of Amino Acid Residues in the P3 Position Directs Substrates to Distinct Catalytic Sites of the Pituitary Multicatalytic Proteinase Complex (Proteasome)

Biochemistry ◽  
1994 ◽  
Vol 33 (21) ◽  
pp. 6483-6489 ◽  
Author(s):  
Christopher Cardozo ◽  
Alexander Vinitsky ◽  
Charlene Michaud ◽  
Marian Orlowski
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Multicatalytic Proteinase ◽  
Catalytic Sites ◽  
Multicatalytic Proteinase Complex

scholarly journals Leupeptin-binding site(s) in the mammalian multicatalytic proteinase complex

1993 ◽  
Vol 289 (1) ◽  
pp. 45-48 ◽  
Author(s):  
P J Savory ◽  
A J Rivett
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Antigen Processing ◽  
Basic Amino Acid ◽  
Amino Acid Residues ◽  
Thiol Groups ◽  
Multicatalytic Proteinase ◽  
Different Types ◽  
Proteolytic Activities ◽  
Multicatalytic Proteinase Complex

The multicatalytic proteinase (MCP) complex is a major nonlysosomal proteinase which plays an important role in non-lysosomal pathways of protein degradation and which has recently been implicated in antigen processing. The mammalian MCP complex is composed of more than 20 different types of polypeptide, but it is not yet clear which of these components are responsible for its proteolytic activities. The complex has at least three distinct types of proteolytic activity. One of these, the so-called ‘trypsin-like’ activity, which involves cleavage on the carboxy side of basic amino acid residues, can be selectively and completely inhibited by peptidyl arginine aldehydes (such as leupeptin and antipain), and is also the most sensitive to inhibition by thiol-reactive reagents. In the present study N-[ethyl-1-14C]ethylmaleimide has been used to specifically label thiol groups protected by leupeptin binding. The results suggest that one or two polypeptide components within the complex can be protected against modification by N-ethylmaleimide. These components may be responsible for the ‘trypsin-like’ activity of the complex or may be adjacent to the catalytic component(s) and play an important role in substrate binding.

scholarly journals Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes

1993 ◽  
Vol 296 (3) ◽  
pp. 601-605 ◽  
Author(s):  
P J Savory ◽  
H Djaballah ◽  
H Angliker ◽  
E Shaw ◽  
A J Rivett
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
The Other ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Hydrophobic Amino Acid ◽  
Activity Inhibition ◽  
Peptide Hydrolase ◽  
Catalytic Sites ◽  
Affinity Labelling

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as ‘chymotrypsin-like’, ‘trypsin-like’ and ‘peptidylglutamyl-peptide hydrolase’ activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit ‘chymotrypsin-like’ activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of ‘peptidylglutamyl-peptide hydrolase’ activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

scholarly journals Conserved Structural Regions Involved in the Catalytic Mechanism of Escherichia coli K-12 WaaO (RfaI)

1998 ◽  
Vol 180 (20) ◽  
pp. 5313-5318 ◽  
Author(s):  
Keigo Shibayama ◽  
Shinji Ohsuka ◽  
Toshihiko Tanaka ◽  
Yoshichika Arakawa ◽  
Michio Ohta
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Glycosidic Linkage ◽  
Hydrophobic Cluster Analysis ◽  
E Coli ◽  
Catalytic Sites ◽  
K 12

ABSTRACT Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.

Substrate Recognition by Vitamin K-Dependent Carboxylase

1987 ◽  
Vol 57 (01) ◽  
pp. 017-019 ◽  
Author(s):  
Magda M W Ulrich ◽  
Berry A M Soute ◽  
L Johan M van Haarlem ◽  
Cees Vermeer
Keyword(s):  
Amino Acid ◽  
Vitamin K ◽  
Substrate Recognition ◽  
Bovine Liver ◽  
Amino Acid Residues

SummaryDecarboxylated osteocalcins were prepared and purified from bovine, chicken, human and monkey bones and assayed for their ability to serve as a substrate for vitamin K-dependent carboxylase from bovine liver. Substantial differences were observed, especially between bovine and monkey d-osteocalcin. Since these substrates differ only in their amino acid residues 3 and 4, it seems that these residues play a role in the recognition of a substrate by hepatic carboxylase.

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