ICE processing and kinetic mechanism.

D A Giegel

Home/Archive/PubMed

PubMed · 9015749

ICE processing and kinetic mechanism.

D A Giegel

View original source ↗Export metadata ↓

Abstract

Interleukin-1 beta converting enzyme (ICE) has been the focus of major scientific efforts to discover pharmaceutically effective inhibitors. Little is known about the rates of the individual steps in catalysis. We report here that the rates of the two individual chemical steps in catalysis (acylation and deacylation) are each partially rate-limiting. This keeps the overall rate of the reaction less than 3% of the rate of the reaction for papain with its optimized substrate. Eight human ICE-like proteases have been published to date. They have levels of sequence identity that range from around 30% to greater than 50% throughout the full lengths of the proteins. This degree of relatedness increases when only the active domains are compared. This indicates that the greatest variability between family members occurs in their N-terminal prodomains. We propose several possibilities for the role for these prodomains in the regulation of enzyme processing.

Explore related subjects

Acylation Binding Sites Caspase 1 Cysteine Endopeptidases Enzyme Activation Hydroxylamine Hydroxylamines Kinetics Models, Biological Papain Protein Conformation Recombinant Proteins Journal Article

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

D A Giegel. 1997. ICE processing and kinetic mechanism.. https://pubmed.ncbi.nlm.nih.gov/9015749/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate.

Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained.

Acylation↗

PubMed2007

Dual lipid modification of Arabidopsis Ggamma-subunits is required for efficient plasma membrane targeting.

Posttranslational lipid modifications are important for proper localization of many proteins in eukaryotic cells. However, the functional interrelationships between lipid modification processes in plants remain unclear. Here we demonstrate that the two heterotrimeric G-protein gamma-subunits from Arabidopsis (Arabidopsis thaliana), AGG1 and AGG2, are prenylated, and AGG2 is S-acylated. In wild type, enhanced yellow fluorescent protein-fused AGG1 and AGG2 are associated with plasma membranes, with AGG1 associated with internal membranes as well. Both can be prenylated by either protein geranylgeranyltransferase I (PGGT-I) or protein farnesyltransferase (PFT). Their membrane localization is intact in mutants lacking PFT activity and largely intact in mutants lacking PGGT-I activity but is disrupted in mutants lacking both PFT and PGGT-I activity. Unlike in mammals, Arabidopsis Ggammas do not rely on functional Galpha for membrane targeting. Mutation of the sixth to last cysteine, the putative S-acylation acceptor site, causes a dramatic change in AGG2 but not AGG1 localization pattern, suggesting S-acylation serves as an important additional signal for AGG2 to be targeted to the plasma membrane. Domain-swapping experiments suggest that a short charged sequence at the AGG2 C terminus contributes to AGG2's efficient membrane targeting compared to AGG1. Our data show the large degree to which PFT and PGGT-I can compensate for each other in plants and suggest that differential lipid modification plays an important regulatory role in plant protein localization.

Acylation↗

PubMed2007

New bradykinin analogues acylated on the N-terminus: effect on rat uterus and blood pressure.

Our previous studies suggested that acylation of the N-terminus of several known B2 antagonists with various kinds of bulky acyl groups consistently improved their antagonistic potency in rat blood pressure assay. On the other hand, our earlier observations also seemed to suggest that the effects of acylation on the contractility of isolated rat uterus depended substantially on the chemical character of the acyl group, as we observed that this modification might either change the range of antagonism or even transform it into agonism. Bearing all this in mind, we decided to synthesize seven new analogues of bradykinin by N-terminal acylation with various acyl groups of a moderately potent B2 antagonist, previously synthesized by Stewart's group, D-Arg-Arg-Pro-Hyp-Gly-Thr-Ser-D-Phe-Thi-Arg. The analogues were tested in vitro for their blood pressure-lowering and uterotonic activities. The modifications either preserved or increased the antagonistic potency in the rat blood pressure test. On the other hand, all seven substituents negatively influenced the interaction with the rat uterine receptors. Our results may be helpful for designing new B2 agonists and antagonists.

Acylation↗