Enzymes are extremely useful and effective in many biochemical reactions but only at the right time and place. Enzyme activity is regulated in five different ways:
Allosteric control:Allosteric enzymes contain distinct regulatory sites and multiple functional sites. The protein is significantly controlled when small signal molecules bind to these regulatory sites. Also allosteric enzymes show cooperativity, which means that activity at one functional site will affect the other functional site as well.
Multiple Forms of Enzymes: Isoenzymes or Isozymes are homologous enzymes in an organism that catalyze the same reaction but are a little bit different in their structure, Km and Vmax values, and regulatory properties. Isozymes allow a reaction to be regulated at distinct locations or times.
Reversible Covalent Modification: The catalytic properties of enzymes can be altered by a covalent binding of a modifying group, most commonly to a phosphoryl group. Usually ATP will serve as a donor for these reactions.
Proteolytic Activation: The other regulatory mechanisms mentioned so far can freely change between active and inactive states. However in proteolytic activation, the enzyme is irreversibly converted into from an inactive enzyme to an active one. These enzymes are activated by hydrolysis of a few peptide bonds. Also hydrolysis of an enzyme precursor such as zymogens or proenzymes can also activate the enzyme.
Controlling the Amount of Enzyme Present: Enzyme activity can also be regulated by adjusting the amount of enzymes present. This method of regulation usually takes place during gene transcription.
The first step in the biosynthesis of pyrimidines, the condensation of asparatate and carbamoyl phosphate to form N-carbamoylaspartate and orthophosphate is catalyzed by an allosteric enzyme, aspartate transcarbamoylase or ATCase.
John Gerhart and Arthur Pardee found that ATCase is inhibited by its own final product, the pyrimidine CTP. Therefore as the concentration of CTP increases, the reaction with ATCase slows down. This is a negative feedback loop, or negative inhibition. Allosterically regulated enzymes don't follow Michaelis-Menten Kinetics.
As illustrated here the graph of ATCase kinetics is sigmoidal instead of the Michaelis-Menten hyperbolic shape.
ATCase Consists of Separable Catalytic and Regulatory Subunits ATCase can be separated into regulatory and catalytic substrates by treatment with compounds such as p-hydroxymercuribenzoate. This is evidence that ATCase has distinct regulatory and catalytic sites. John Gerhart and Howard Schachman were the ones to carry out this study. The subunits can then be separate through ion-exchange chromatography or by centrifugation in a sucrose density gradient.
The larger subunit is the Catalytic subunit. This subunit is catalytic as implied by the name but is unresponsive to CTP and does not show sigmoidal kinetics. The other subunit, the regulatory subunit has no catalytic activity but binds to CTP. Therefore ATCase is composed of catalytic and regulatory subunits.
Similar to Hemoglobin, ATCase exists in a T-state and R-state. The T-state is the less active state while the R-state is the active state. CTP inhibits ATCase by binding to the regulatory sites stabilizing the T-state. ATP can also bind to the same sites, but does not stabilize the T-state. Therefore ATP competes with CTP for the sites. ATP is an allosteric activator that binds to the regulatory subunit. ATP as well as CTP are referred to as "heterotropic effects" on a allosteric enzyme such as ATCase. ATP is an allosteric activator of aspartate transcarbamolyase because it stabilizes the R-state of ATCase, effecting neighboring subunits by making it easier for substrate to bind.The increase of the concentration of ATP has two potential explanations. First being, at high concentrations of ATP signals a high concentration of purine and pyrimidine. second, a high concentration of ATP conveys that a source of energy is available for mRNA synthesis and DNA replication follow by the synthesis of pyrimidines needed for these processes.
Enzymes that differ in amino acid sequence but catalyze the same reaction are called isoenzymes. Generally, Isoenzyme have different Km and respond to different regulatory molecules. Different genes encode Isozymes. Isozymes allows specific adjustments to be made to metabolism to accommodate the needs of a tissue or developmental stage.
The activity of an enzyme can be modified by covalently bonding a molecule to it. Most modifications are reversible. The most common covalent modifications are Phosphorylation and dephosphorylation.
Phosphorylation: Almost every metabolic process in eukaryotic cells are regulated by phosphorylation. As much as 30% of eukaryotic proteins are phosphorylated. Phosphoryl groups are usually donated by ATP. The gamma terminal phosphoryl group of ATP is transferred to an amino acid. The amino acid acceptor always has a hydroxyl group in the side chain. Kinases transfers the phosphoryl groups and Protein phosphatases reverses the process. However phosphorylation and dephosphorylation are not reverse reactions of one another. Each reaction is almost irreversible under normal physiological conditions. Phosphorylation will only take place through a specific protein kinase using an ATP and depphosphorylation will only occur through phosphatase.
- Adds two negative charges
- Forms 2 or 3 hydrogen bonds
- Phosphorylation is reversible
- Kinetics can be adjusted to physiological process
- Amplifies sign
- ATP coordinates signaling with bioenergetics
Common Covalent Modifications of Protein Activity
- Donates ATP to glygogen phosphorylase which functions in glucose homeostasis and energy transduction
- Donates Acetyl CoA to histones which functions in DNA packing and transcription
- Donates Myristoyl CoA to Src which functions in signal transduction
- ADP ribosylation
- Donates NAD+ to RNA polymerase which functions in transcription
- Donates Farnesyl Pyrophosphate to Ras which functions in signal transduction
- γ-Carboxylation Sulfation
- Donates HCO3- and 3'-Phosphoadenosine-5'-phosphosulfate to fibrinogen which functions in blood-clot formation
- Donates Ubiquitin to cyclin which functions in the control of cell cycle
Many enzymes are inactive until one or a few specific peptide bonds are cleaved. The enzyme exists initially as an inactive precursor called zymogen or proenzyme. Proteolytic cleavage does not need ATP for energy and only occurs once in the life of the enzyme. Some examples of enzymes and biochemical processes that are activated by proteolytic cleavage are:
The digestive system that hydrolyze proteins are initially made as zymogens in the stomach and pancreas
Blood clotting is mediated by a cascade of proteolytic activations
Insulin is derived from the inactive precursor proinsulin
Proteolytic activation of chymotrypsinogen forming an active chymotrypsin is involved in digestive enzymes, blood clotting, protein hormones, and procaspases (a programmed cell death).
Berg, Jeremy M. Tymoczko, John L. Stryer, Lubert. Biochemistry 6th Edition. Copyright 2007, 2002 by W. H. Freeman and Company
Viadiu, Hector. Reversible Covalent Modification. Biochemistry Lecture. Dec. 5, 2012