Related Experiment Video
Updated: Jul 31, 2026
19:16
The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 17, 2010
Cross-linking preserves conformational changes induced in penicillinase by its substrates
The Biochemical Journal
|May 1, 1980
Summary
Bacillus cereus exopenicillinase enzyme conformation changes when bound to substrates like cephalothin or cloxacillin. These conformational changes affect the enzyme
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Exopenicillinase from Bacillus cereus is a key enzyme in penicillin hydrolysis.
- Understanding enzyme structure-function relationships is crucial for drug development and biotechnology.
- Substrate binding can induce conformational changes in enzymes, affecting their stability and activity.
Purpose of the Study:
- To investigate the conformational changes in Bacillus cereus exopenicillinase upon substrate binding.
- To determine how these conformational changes influence the enzyme's susceptibility to inactivation.
- To explore the potential of using cross-linking to probe enzyme-substrate interactions.
Main Methods:
- Cross-linking of exopenicillinase with toluene 2,4-diisocyanate in the presence of cephalothin, cloxacillin, or no substrate.
- Assessing the susceptibility of the resulting enzyme derivatives to inactivation by heat, urea, iodination, and proteolysis.
- Analyzing the correlation between substrate-induced conformational changes and enzyme inactivation rates.
Main Results:
- Cross-linked exopenicillinase derivatives exhibited distinct differences in their resistance to inactivation.
- The presence of cephalothin or cloxacillin during cross-linking altered the enzyme's susceptibility to various inactivation methods.
- These observed differences in inactivation susceptibility correlated with the known effects of these substrates on enzyme conformation.
Conclusions:
- Substrate binding significantly alters the conformation of Bacillus cereus exopenicillinase.
- Conformational changes induced by substrates like cephalothin and cloxacillin directly impact enzyme stability and susceptibility to inactivation.
- Cross-linking in the presence of substrates provides a method to study enzyme conformational dynamics and their functional consequences.
Related Concept Videos
01:49Conserved Binding Sites
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
00:49Ligand Binding and Linkage
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 the...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
01:13Introduction to Mechanisms of Enzyme Catalysis
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 a mild...
01:27Production of Antibiotics
Penicillin, one of the earliest and most widely used antibiotics, is produced industrially by the filamentous fungus Penicillium chrysogenum. Large stirred-tank bioreactors ranging from tens to hundreds of thousands of liters maintain tightly controlled temperature, pH, and dissolved oxygen conditions to support fungal metabolism and maximize antibiotic yield. Penicillin is a secondary metabolite, synthesized primarily during the stationary growth phase, which requires a carefully managed...
01:23Inhibitors of Gram-positive Cell Wall Synthesis
Bacterial cell walls are typically rigid structures composed mainly of peptidoglycan, a mesh-like polymer that provides mechanical strength and maintains cell shape. The synthesis of peptidoglycan is a crucial process in bacterial growth and serves as a primary target for many antibiotics.Mechanism of Action of Beta-Lactam AntibioticsBeta-lactam antibiotics, such as penicillin, inhibit peptidoglycan synthesis in actively growing cells. These antibiotics share a characteristic four-membered...