For an enzyme to exert its effect on a substrate, the substrate must enter the active site of the enzyme to form the enzyme-substrate complex (the first step of the Michaelis-Menton mechanism). In 1890, Emil Fischer proposed a model for how a substrate fits into the active site of an enzyme, known as the lock-and-key model. In this model, the amino acids that make up an enzyme's active site in the unbound state are said to form a shape that exactly matches the shape of the substrate. Thus, the substrate fits into this active site, just as a key fits into a lock whose shape is designed to match the key (Figure 1).
Schematic of the lock-and-key model. The substrate has a shape that fits exactly in the active site of the unbound enzyme, forming the enzyme-substrate complex.
This model is quite useful for visualizing how the enzyme-substrate complex is formed as a crucial step in catalyzing biological reactions. However, the active sites of many enzymes do not have a shape in the unbound form that exactly matches the shape of the substrate. The shape of the active site changes when the substrate binds to the enzyme, creating a shape into which the substrate fits. This process, known as the induced-fit model, was described by Daniel E. Koshland, Jr. in 1958. Today, this concept is typically used to describe the substrate-binding behavior of many different enzymes.
In the human body, proteins are essential molecules in organisms and have a multitude of functions ranging from providing tensile strength to bones and tendons to providing storage and transportation of necessary substances such as O2 and iron throughout the body. Hence, within the body's cells, proteins from foods must first be separated into their constituent amino acids. Then these amino acids are used to construct the proteins needed by our body.
To break down a protein into its constituent amino acids, the cell uses a hydrolysis reaction. The protein reacts with a water molecule to produce an amino acid and a new smaller protein. The enzyme carboxypeptidase A is secreted by the pancreas and is used to speed up this hydrolysis reaction. As seen in Figure 2, this enzyme consists of a single chain of 307 amino acids. It assumes a compact, globular shape containing regions of both a helices and b pleated sheets. This globular shape contains a region resembling a pocket, where a substrate can fit. This region is the active site of the enzyme.
Carboxypeptidase A is a good illustration of the induced-fit theory, because the active site changes appreciably when the substrate binds. Figures 2 and 3 show three-dimensional representations of the carboxylase protein with and without a bound substrate. Note how the active site changes shape when it is complexed with a substrate. As the protein substrate binds to carboxypeptidase, the active site closes in around it. Hydrolysis of the peptide bond is most likely to occur if the terminal residue has an aromatic or bulky hydrocarbon side chain. A zinc ion (Zn2+) is tightly bound near the active site and assists in catalysis. Three hydrogen bonding and electrostatic interactions are critical for the enzyme to recognize the terminal amino acid in the peptide chain. The intermediate is stabilized by interactions with Zn2+ and the carboxypeptidase molecule. The last step is a proton transfer and cleavage of the peptide bond. This entire process requires considerable mobility of the carboxypeptidase A protein itself.
This is a molecular model of the unbound carboxypeptidase A enzyme. The cpk, or space-filled, representation of atoms is used here to show the approximate volume and shape of the active site. Note the zinc ion (magenta) in the pocket of the active site. Three amino acids located near the active site (Arg 145, Tyr 248, and Glu 270) are labeled.
This is a cpk representation of carboxypeptidase A with a substrate (turquoise) bound in the active site. The active site is in the induced conformation. The same three amino acids (Arg 145, Tyr 248, and Glu 270) are labeled to demonstrate the shape change.
Note: Coordinates for Figures 2 and 3 are from x-ray crystallographic data.
1. A researcher has obtained high-quality three-dimensional images of the active site of the unbound carboxypeptidase A molecule and of the compound l-phenyl lactate. Upon examining these images, she notes that the shape of the active site does not match the shape of l-phenyl lactate. She concludes that l-phenyl lactate cannot form a substrate complex with carboxypeptidase A, because it does not fit into the active site. Do you agree with this researcher's conclusion? Briefly explain your answer.
2. Below is a schematic of a protein where R and R' each represent one of 20 possible substituents:
a. In the hydrolysis reaction described in the tutorial, the Zn2+ ion bonds to the carbonyl group (C=O) of the penultimate amino acid of the substrate. To which atom in the carbonyl group does the Zn2+ ion bond? What type of interaction occurs between the Zn2+ ion and the carbonyl group?
b. The structures of Arginine and Tyrosine are shown below. The residues Arg 145 and Tyr 248 from the enzyme form hydrogen bonds with the protein. In the terminal amino-acid fragment, to what atoms do the side chains of these residues form hydrogen bonds?
3. The active site of the enzyme carboxypeptidase A has a hydrophobic pocket where the side chain of the terminal amino acid of the substrate protein resides. What type of side chain must this amino acid contain and what type of interaction occurs between the side chain and this hydrophobic pocket?
To view the molecules interactively, please use RASMOL. To download the pdb files for viewing and rotating the molecules shown above, please click on the appropriate name below. (Note: if you want to view multiple molecules simultaneously, please download each file (.pdb) and save on your computer. Then open RASMOL outside of Netscape.)
Greenblatt, H.M. et al.. "Carboxypeptidase A: Native, zinc-removed, and mercury-replaced forms." (1997).To be published. PDB coordinates as "Structure of Carboxypeptidase," Brookhaven Protein Data Bank.
Stryer, Lupert. Biochemistry. 4th ed., W.H. Freeman and Co., New York, 1995, p. 218-222.
Teplyakov, A. et al. "The high resolution crystal structure of the complex between carboxypeptidase A and l-phenyl lactate." (1994)To be published. PDB coordinates, Brookhaven Protein Data Bank.
Zundahl, S. Chemical Principles. 3rd ed., Houghton Mifflin Co., New York, 1998, p. 710-711, 1019-1020.
The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education program, Grant HHMI#71192-502004 to Washington University.