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

ABSTRACT: 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.

SUBMITTER: Savory PJ

PROVIDER: S-EPMC1132128 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Project description:The activity of multicatalytic proteinase against synthetic substrates and the kinetics of its inhibition by a range of class-specific inhibitors have been investigated. The enzyme was found to have a broader pH activity profile than previously noted, being active against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin optimally at pH 4.5 and against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin optimally at pH 10.5. Neither activity was inhibited by the class-specific inhibitors 1,10-phenanthroline, EDTA, pepstatin, di-isopropyl fluorophosphate, peptidyl chloromethanes, peptidyl diazomethanes or L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64), indicating that the enzyme is not a typical metallo-, aspartic, serine or cysteine proteinase. Inhibition by HgCl2, iodoacetamide and N-ethylmaleimide suggests that free thiols are necessary for the enzyme to maintain activity, but that these thiols are not particularly reactive as is the case for cysteine proteinases of the papain superfamily. The peptidyl aldehydes chymostatin and leupeptin were found to be reversible inhibitors of multicatalytic proteinase. Chymostatin inhibited activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin at pH 4.5 (Ki 160 +/- 22 microM) whereas leupeptin (200 microM) was not inhibitory. Inhibition of activity against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin by these compounds was more complex, in that they behaved as slow tight-binding inhibitors. kon values were determined to be 12 +/- 2 M-1.s-1 and 1290 +/- 125 M-1.s-1 for chymostatin and leupeptin, respectively. The upper limit for Ki values for these two inhibitors was estimated as 5 +/- 1.5 microM and 25 +/- 5 nM, respectively. The different inhibition characteristics for each substrate were also apparent at an intermediate pH of 8.5, showing that the two activities are distinct. Dichloroisocoumarin, a mechanism-based inhibitor of serine proteinases, did inhibit activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin with a rate constant of 250 M-1.s-1, suggesting that multicatalytic proteinase is an atypical serine proteinase.

Project description:The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.

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

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