Related Experiment Video
Updated: Jul 11, 2026
08:59
Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
Probing steric and hydrophobic effects on enzyme-substrate interactions by protein engineering
Science (New York, N.Y.)
|August 8, 1986
Summary
Protein engineering of subtilisin revealed how steric and hydrophobic effects influence substrate specificity. Hydrophobic substitutions enhanced catalytic efficiency for small substrates, while steric hindrance from larger molecules decreased it significantly.
Area of Science:
- Enzymology
- Protein Engineering
- Biochemistry
Background:
- Subtilisin is a protease with broad specificity and a large, hydrophobic substrate-binding cleft.
- Understanding enzyme-substrate interactions is crucial for enzyme engineering.
Purpose of the Study:
- To investigate the impact of steric and hydrophobic effects on subtilisin's substrate specificity.
- To elucidate the role of amino acid substitutions at position 166 in the substrate-binding cleft.
Main Methods:
- Protein engineering using cassette mutagenesis to replace glycine at position 166 with 12 different amino acids.
- Characterization of mutant subtilisin enzymes' specificity and catalytic efficiency (kcat/Km).
Main Results:
- Hydrophobic substitutions at position 166 increased catalytic efficiency (up to 16-fold) for small, hydrophobic substrates.
- Enlarging the substrate or the binding cleft side chain led to steric hindrance, causing drastic drops in catalytic efficiency (up to 5000-fold).
Conclusions:
- Substrate size and hydrophobicity, along with binding cleft volume, are critical determinants of subtilisin's substrate specificity.
- Strategic amino acid modifications can optimize enzyme activity for specific substrates, but steric limitations must be considered.
Related Concept Videos
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...
01:13Induced-fit Model
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 characteristics of...
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical characteristics of...
02:34Enzymes
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...
01:08Allosteric Regulation
Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
01:19Allosteric Proteins-ATCase
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 pathway,...
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 pathway,...
02:04Protein-protein Interfaces
Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide...