Related Experiment Video
Updated: May 15, 2025
09:47
The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes
Published on: May 25, 2018
6.7K
Structural basis for substrate selectivity by site-one protease revealed by studies with a small-molecule inhibitor
Ashley V Bullington1, Ilaria Micallo2,3,4, Bilkish Bajaj1
1Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390.
Summary
Site-one protease (S1P) activity, crucial for lipogenesis and ER stress, was investigated using the inhibitor PF-429242. Structural analysis revealed how S1P recognizes substrates, enabling the design of resistant S1P variants.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Site-one protease (S1P) is essential for activating effector proteins in the Golgi, regulating key cellular pathways like lipogenesis and ER stress.
- S1P maturation involves autocatalysis in the ER and cofactor SPRING, with its activity spatially controlled for signaling.
- Understanding S1P substrate recognition is critical for elucidating these signaling pathways.
Purpose of the Study:
- To investigate the structural basis of substrate recognition by the S1P/SPRING complex.
- To utilize the competitive inhibitor PF-429242 to probe S1P's active site and substrate binding pocket.
- To design and validate S1P variants with altered substrate recognition properties.
Main Methods:
- Determined the crystal structure of the S1P/SPRING complex bound to the inhibitor PF-429242.
- Performed structural analysis to identify key residues involved in substrate binding.
- Designed and tested an S1P mutant (I308A) for resistance to PF-429242 in biochemical and cell-based assays.
Main Results:
- PF-429242 binds to the S1P active site in the same pocket as the substrate's P4 Arg residue.
- Structural data suggests S1P undergoes a conformational change to accommodate the P2 Leu/Ile/Val residue of substrates.
- The I308A mutation conferred resistance to PF-429242, indicating reduced steric hindrance at the P2 position.
Conclusions:
- The study reveals the structural basis for S1P's substrate selectivity, particularly at the P2 position.
- Findings provide insights into the mechanism of S1P-mediated proteolysis and cofactor SPRING interaction.
- This work offers a foundation for the rational design of novel and improved S1P inhibitors.
Related Concept Videos
02:34Enzymes
80.2K
Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
80.2K
01:30Enzyme Inhibition
77.5K
Inhibitors are molecules that reduce enzyme activity by binding to the enzyme. In a normally functioning cell, enzymes are regulated by a variety of inhibitors. Drugs and other toxins can also inhibit enzymes. Some inhibitors bind to the enzyme’s active site, while others inhibit enzymatic activity by binding to other sites on the protein structure.
77.5K
01:13Induced-fit Model
79.9K
Most chemical reactions in cells require enzymes—biological catalysts that speed up the reaction without being consumed or permanently changed. They reduce the activation energy needed to convert the reactants into products. Enzymes are proteins, that usually work by binding to a substrate—a reactant molecule that they act upon.
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
79.9K
01:19Allosteric Proteins-ATCase
5.6K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
5.6K
00:49Ligand Binding and Linkage
4.7K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
4.7K
01:13Introduction to Mechanisms of Enzyme Catalysis
7.8K
For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes...
7.8K