In the human body, thousands of diverse enzymes are regulated to fulfill their individual functions without waste of dietary components. Thus, with changes in our physiologic state, time of eating, environment, diet, or age, the rates of some enzymes must increase and those of others decrease. In this chapter, we describe the mechanisms for regulating enzyme activity and the strategies employed to regulate the metabolic pathways in which they reside.
Regulation Matches Function. Changes in the rate of a metabolic pathway occur because at least one enzyme in that pathway, the regulatory enzyme, has been activated or inhibited, or the amount of enzyme has increased or decreased. Regulatory enzymes usually catalyze the rate-limiting, or slowest, step in the pathway, so that increasing or decreasing their rate changes the rate of the entire pathway (Fig. 9.1). The mechanisms used to regulate the rate-limiting enzyme in a pathway reflect the function of the pathway.
Substrate Concentration. The rate of all enzymes depends on substrate concentration. Enzymes exhibit saturation kinetics; their rate increases with increasing substrate concentration [S], but it reaches a maximum velocity (Vmax) when the enzyme is saturated with substrate. For many enzymes, the Michaelis–Menten equation describes the relationship between vi (the initial velocity of a reaction), [S], Vmax, and the Km (the substrate concentration at which vi = ½Vmax).
Reversible Inhibition. Enzymes are reversibly inhibited by structural analogs and products. These inhibitors are classified as competitive, noncompetitive, or uncompetitive, depending on their effect on formation of the enzyme–substrate complex.
Allosteric Enzymes. Allosteric activators or inhibitors are compounds that bind at sites other than the active catalytic site and regulate the enzyme through conformational changes that affect the catalytic site.
Covalent Modification. Enzyme activity may also be regulated by a covalent modification, such as phosphorylation of a serine, threonine, or tyrosine residue by a protein kinase.
Protein–Protein Interactions. Enzyme activity can be modulated through the reversible binding of a modulator protein, such as Ca2+–calmodulin. Monomeric G-proteins (guanosine triphosphate [GTP]–binding proteins) activate target proteins through reversible binding.
Zymogen Cleavage. Some enzymes are synthesized as inactive precursors, called zymogens, that are activated by proteolysis (e.g., the digestive enzyme chymotrypsin).
Changes in Enzyme Concentration. The concentration of an enzyme can be regulated by changes in the rate of enzyme synthesis (e.g., induction of gene transcription) or the rate of degradation.
Regulation of Metabolic Pathways. The regulatory mechanisms for the rate-limiting enzyme of a pathway always reflect the function of the pathway in a particular tissue. In feedback regulation, the end product of a pathway directly or indirectly controls its own rate of synthesis; in feed-forward regulation, the substrate controls the rate of the pathway. Biosynthetic and degradative pathways are controlled through different but complementary regulation. Pathways are also regulated through compartmentation of enzymes unique to specific pathways.
THE WAITING ROOM
Al M. is a 44-year-old man who has suffered from alcohol use disorder for the past 5 years. He was recently admitted to the hospital for congestive heart failure (see Chapter 8). After being released from the hospital, he continued to drink. One night he arrived at a friend’s house at 7:00 P.M. Between his arrival and 11:00 P.M., he drank four beers and five martinis (for a total ethanol consumption of 9.5 oz). His friends encouraged him to stay an additional hour to try and sober up. Nevertheless, he ran his car off the road on his way home. He was taken to the emergency department of the local hospital and arrested for driving under the influence of alcohol. His blood alcohol concentration at the time of his arrest was 240 mg/dL, compared with the legal limit of ethanol for driving of 80 mg/dL (0.08% blood alcohol).
Ann R., a 23-year-old woman, 5 ft 7 in tall, is being treated for anorexia nervosa (see Chapters 1 through 3). She has been gaining weight and is now back to 99 lb from a low of 85 lb. Her blood glucose is within the low normal range (fasting blood glucose of 72 mg/dL, compared with a normal range of 70 to 100 mg/dL). She complains to her physician that she feels tired when she jogs, and she is concerned that the “extra weight” she has gained is slowing her down. Regulation of various metabolic pathways is critical as she gains weight and strives to restore normal activity levels.
I. General Overview
Although the regulation of metabolic pathways is an exceedingly complex subject, dealt with in most of the subsequent chapters of this text, a number of common themes are involved. Physiologic regulation of a metabolic pathway depends on the ability to alter flux through the pathway by activating the enzyme catalyzing the rate-limiting step in the pathway (see Fig. 9.1). The type of regulation employed always reflects the function of the pathway and the need for that pathway in a particular tissue or cell type. Pathways that produce a necessary product are usually feedback-regulated through a mechanism that involves concentration of product (e.g., allosteric inhibition or induction/repression of enzyme synthesis), either directly or indirectly. The concentration of the product signals when enough of the product has been synthesized. Storage and toxic disposal pathways are usually regulated directly or indirectly through a feed-forward mechanism that reflects the availability of precursor. Regulatory enzymes are often tissue-specific isozymes whose properties reflect the different functions of a pathway in particular tissues. Pathways are also regulated through compartmentation, the collection of enzymes with a common function within a particular organelle or at a specific site in the cell.
The mechanisms employed to regulate enzymes have been organized into three general categories: regulation by compounds that bind reversibly in the active site (including the dependence of velocity on substrate concentration and product levels), regulation by changing the conformation of the active site (including allosteric regulators, covalent modification, protein–protein interactions, and zymogen cleavage), and regulation by changing the concentration of enzyme (enzyme synthesis and degradation). We will generally be using the pathways of fuel oxidation to illustrate the role of various mechanisms of enzyme regulation in metabolic pathways (see Chapters 1 through 3 for a general overview of these pathways).
A. Velocity and Substrate Concentration
The velocity (rate of formation of product per unit time) of all enzymes is dependent on the concentration of substrate. This dependence is reflected in conditions such as starvation, in which a number of pathways are deprived of substrate. In contrast, storage pathways (e.g., glucose conversion to glycogen in the liver) and toxic-waste disposal pathways (e.g., the urea cycle, which prevents NH3 toxicity by converting NH3 to urea) are normally regulated to speed up when more substrate is available. In the following sections, we use the Michaelis–Menten equation to describe the response of an enzyme to changes in substrate concentration, and we use glucokinase (GK) to illustrate the role of substrate supply in regulation of enzyme activity.
1. The Michaelis–Menten Equation
The equations of enzyme kinetics provide a quantitative way of describing the dependence of enzyme rate on substrate concentration. The simplest of these equations, the Michaelis–Menten equation, relates the initial velocity (vi) to the concentration of substrate [S] (the brackets denote concentration) and the two parameters Km and Vmax (Equation 9.1). The Vmax of the enzyme is the maximal velocity that can be achieved at an infinite concentration of substrate, and the Km of the enzyme for a substrate is the concentration of substrate required to reach ½Vmax. The Michaelis–Menten model of enzyme kinetics applies to a simple reaction in which the enzyme and substrate form an enzyme–substrate complex (ES) that can dissociate back to the free enzyme and substrate. The initial velocity of product formation, vi, is proportional to the concentration of enzyme–substrate complexes [ES]. As substrate concentration is increased, the concentration of ES complexes increases, and the reaction rate increases proportionately. The total amount of enzyme present is represented by Et.
For the reaction
the Michaelis–Menten equation is given by
where Km = (k2 + k3)/k1
and Vmax = k3 [Et]
The graph of the Michaelis–Menten equation (vi as a function of substrate concentration) is a rectangular hyperbola that approaches a finite limit, Vmax, as the fraction of total enzyme present as ES complex increases (Fig. 9.2). At a hypothetical infinitely high substrate concentration, all of the enzyme molecules contain bound substrate, and the reaction rate is at Vmax. The approach to the finite limit of Vmax is called saturation kinetics, because velocity cannot increase any further once the enzyme is saturated with substrate. Saturation kinetics is a characteristic property of all rate processes that depend on the binding of a compound to a protein.
The Km of the enzyme for a substrate is defined as the concentration of substrate at which vi equals ½Vmax. The velocity of an enzyme is most sensitive to changes in substrate concentration over a concentration range below its Km (see Fig. 9.2). As an example, at substrate concentrations less than one-tenth of the Km, a doubling of substrate concentration nearly doubles the velocity of the reaction; at substrate concentrations 10 times the Km, doubling the substrate concentration has little effect on the velocity.
The Km of an enzyme for a substrate is related to the dissociation constant, Kd, which is the rate of substrate release divided by the rate of substrate binding (k2/k1). For example, a genetic mutation that decreases the rate of substrate binding to the enzyme decreases the affinity of the enzyme for the substrate and increases the Kd and Km of the enzyme for that substrate. The higher the Km, the higher is the substrate concentration required to reach ½Vmax.
2. The Lineweaver–Burk Transformation
The Km and Vmax for an enzyme can be visually determined from a plot of 1/vi versus 1/S called a Lineweaver–Burk or a double-reciprocal plot. The reciprocal of both sides of the Michaelis–Menten equation generates an equation that has the form of a straight line, y = mx + b (Fig. 9.3). Km and Vmax are equal to the reciprocals of the intercepts on the abscissa and ordinate, respectively. Although double-reciprocal plots are often used to illustrate certain features of enzyme reactions, they are not used directly for the determination of Km and Vmax values by researchers.
3. Hexokinase Isozymes Have Different Km Values for Glucose
A comparison between the isozymes of hexokinase (HK) found in red blood cells and in the liver illustrates the significance of the Km of an enzyme for its substrate. HK catalyzes the first step in glucose metabolism in most cells—the transfer of a phosphate from adenosine triphosphate (ATP) to glucose to form glucose 6-phosphate (glucose 6-P). Glucose 6-P may then be metabolized in glycolysis, which generates energy in the form of ATP, or it can be converted to glycogen, a storage polymer of glucose. Hexokinase I, the isozyme in red blood cells (erythrocytes), has a Km for glucose of approximately 0.05 mM (Fig. 9.4). The isozyme of HK called glucokinase (GK), which is found in the liver and pancreas, has a much higher Km of approximately 5 to 6 mM. The red blood cell is totally dependent on glucose metabolism to meet its needs for ATP. At the low Km of the erythrocyte HK, blood glucose could fall drastically below its normal fasting level of approximately 5 mM, and the red blood cell could still phosphorylate glucose at rates near Vmax. The liver, however, stores large amounts of “excess” glucose as glycogen or converts it to fat. Because GK has a Km of approximately 5 mM, the rate of glucose phosphorylation in the liver will tend to increase as blood glucose increases after a high-carbohydrate meal and decrease as blood glucose levels fall. The high Km of hepatic GK thus promotes the storage of glucose as liver glycogen or as fat, but only when glucose is in excess supply.
The rate of a reaction is directly proportional to the concentration of enzyme; if you double the amount of enzyme, you will double the amount of product produced per minute, whether you are at low or at saturating concentrations of substrate. This important relationship between velocity and enzyme concentration is not immediately apparent in the Michaelis–Menten equation because the concentration of total enzyme present (Et) has been incorporated into the term Vmax (i.e., Vmax is equal to the rate constant k3 × Et). However, Vmax is most often expressed as product produced per minute per milligram of enzyme and is meant to reflect a property of the enzyme that is not dependent on its concentration.
5. Multisubstrate Reactions
Most enzymes have more than one substrate, and the substrate-binding sites overlap in the catalytic (active) site. When an enzyme has more than one substrate, the sequence of substrate binding and product release affect the rate equation. As a consequence, an apparent value of Km (Km,app) depends on the concentration of cosubstrate or product present.
6. Rates of Enzyme-Catalyzed Reactions in the Cell
Equations for the initial velocity of an enzyme-catalyzed reaction, such as the Michaelis–Menten equation, can provide useful parameters for describing or comparing enzymes. However, many multisubstrate enzymes, such as GK, have kinetic patterns that do not fit the Michaelis–Menten model (or do so under nonphysiologic conditions). The Michaelis–Menten model is also inapplicable to enzymes present in a higher concentration than their substrates. Nonetheless, the term Km is still used for these enzymes to describe the approximate concentration of substrate at which velocity equals ½Vmax.
B. Reversible Inhibition within the Active Site
One of the ways of altering enzyme activity is through compounds binding in the active site. If these compounds are not part of the normal reaction, they inhibit the enzyme. An inhibitor of an enzyme is defined as a compound that decreases the velocity of the reaction by binding to the enzyme. It is a reversible inhibitor if it is not covalently bound to the enzyme and can dissociate at a significant rate. Reversible inhibitors are generally classified as competitive, noncompetitive, or uncompetitive with respect to their relationship to a substrate of the enzyme. In most reactions, the products of the reaction are reversible inhibitors of the enzyme producing them.
1. Competitive Inhibition
A competitive inhibitor “competes” with a substrate for binding at the enzyme’s substrate-recognition site and therefore is usually a close structural analog of the substrate (Fig. 9.5A). An increase of substrate concentration can overcome competitive inhibition; when the substrate concentration is increased to a sufficiently high level, the substrate-binding sites are occupied by substrate, and inhibitor molecules cannot bind. Competitive inhibitors, therefore, increase the apparent Km of the enzyme (Km,app) because they raise the concentration of substrate necessary to saturate the enzyme. They have no effect on Vmax.
2. Noncompetitive and Uncompetitive Inhibition
If an inhibitor does not compete with a substrate for its binding site, the inhibitor is either a noncompetitive or an uncompetitive inhibitor with respect to that particular substrate (see Fig. 9.5B). Uncompetitive inhibition is almost never encountered in medicine and will not be discussed further. To illustrate noncompetitive inhibition, consider a multisubstrate reaction in which substrates A and B react in the presence of an enzyme to form a product. An inhibitor (NI) that is a structural analog of substrate B would fit into substrate B’s binding site, but the inhibitor would be a noncompetitive inhibitor with regard to the other substrate, substrate A. An increase of A will not prevent the inhibitor from binding to substrate B’s binding site. The inhibitor, in effect, lowers the concentration of the active enzyme and therefore changes the Vmax of the enzyme. If the inhibitor has absolutely no effect on the binding of substrate A, it will not change the Km for A (a pure noncompetitive inhibitor).
Lineweaver–Burk plots provide a good illustration of competitive inhibition and pure noncompetitive inhibition (Fig. 9.6). In competitive inhibition, plots of 1/vi versus 1/[S] at a series of inhibitor concentrations intersect on the y-axis. Thus, at infinite substrate concentration, or 1/[S] = 0, there is no effect of the inhibitor. In pure noncompetitive inhibition, the inhibitor decreases the velocity even when [S] has been extrapolated to an infinite concentration. However, if the inhibitor has no effect on the binding of the substrate, the Km is the same for every concentration of inhibitor, and the lines intersect on the x-axis.