Ординатура / Офтальмология / Английские материалы / Biochemistry of the Eye 2nd edition_Whikehart_2003

Enzymes • 59

Figure 3–4

Graph of enzyme activity (v) vs. substrate concentration [S] showing the difficulty of obtaining a true value of

Vmax, the maximum velocity of the enzyme. The velocity of the enzyme

increases with [S], but never quite

reaches its Vmax. At 1/2 of Vmax, one can measure the affinity of the enzyme for

the substrate (Km).

trations of 1 10–1 and 10–7 M (Stryer, 1988). However, the Vmax and the Km of an enzyme are not usually measured by constructing graphs as

shown in Figure 3–4. This is due to the difficulty in obtaining the value of Vmax as it approaches an asymptotic limit. Another approach to finding Vmax and Km is the use of a double-reciprocal plot such as a LineweaverBurk plot (Figure 3–5). The plot is constructed by using the reciprocal of the Michaelis-Menten equation:

This equation allows one to obtain a straight-line graph having two intercepts (x and y), which give the reciprocal values for Km and Vmax, respectively. In the eye, aldose reductase, an enzyme involved in the formation of sugar cataracts, serves as an example of a Michaelis-Menten enzyme.

Figure 3–5

A Lineweaver-Burk plot of 1/v (reciprocal of enzyme velocity) vs. 1/[S] (reciprocal of the molar concentration of the substrate). Here it is possible to accurately determine both the Vmax and the Km. Enzyme velocities are measured as the concentration of substrate consumed or product formed per unit time at stated conditions of temperature and bathing solutions. The term “units of velocity” (U) may also be used. Note that the x-intercept is a negative value.

60 • Biochemistry of the Eye

Allosteric Enzymes

The name allosteric means “other site” and refers to the fact that the kinetics of these enzymes is influenced by substances bound to the enzyme at locations other than the active site. This can occur in two different ways. Since allosteric enzymes are proteins with quaternary structures (meaning that they consist of more than one polypeptide chain), each chain has its own active site. When only one, or a few, active site(s) are occupied, the affinity of enzyme and substrate is low. In this situation, the velocity of the reaction is also low. As the number of active sites occupied increases, an overall conformational change in the protein enzyme becomes more favorable to increased binding of the substrate to the remaining active sites. Therefore, the velocity of the enzyme increases. The change in the enzyme conformation is described as pro-

Figure 3–6

Schematic diagram of an allosteric enzyme in its T- and R-forms. The enzyme is represented as four polypeptides with an active site (grey circle) in each polypeptide. In the T-form, the substrate has difficulty entering the active site while in the R-form access to the active site is easily gained. This is shown by the narrow and broad openings to the active sites. Inhibitors maintain these enzymes in their T-forms, but activators convert them to their R-forms. Substrates force a conformational change in the enzyme from T-form to R-form after enough substrate has entered the active sites available.

Enzymes • 61

Figure 3–7

The effect of an activator upon the velocity and substrate affinity of an allosteric enzyme. When the activator is bound (left hand curve). The apparent K is decreased (i.e., the affinity is increased) while the velocity is increased.

ceeding from a T-form (T = tense) to an R-form (R = relaxed). A second way that the kinetics is influenced is with the binding of activator substances that occupy sites on the enzyme away from the active sites themselves. In this way, such an enzyme is also induced to change from its T-form to its R-form at much lower substrate concentrations. This is a biological way of jump-starting an enzyme to high velocities at lower substrate concentrations. Figure 3–6 shows a hypothetical enzyme in the T- and R-forms when bound to activators or multiple substrates (inhibitors, also shown, are described later).

Figure 3–7 indicates a graph of velocity versus [S] concentration for an allosteric enzyme without (on the right) and with (on the left) bound activator substances. Note that with such enzymes Km becomes known as

Kapparent (also known as Kapp or K0.5) since the K value with allosteric enzymes is dependent upon activators as well. In the eye, sodium-

potassium activated ATPase, whose role is of special significance in the cornea and the ciliary body, serves as an example of an allosteric enzyme. When one attempts to make a Lineweaver-Burk plot with allosteric enzymes, however, the line becomes nonlinear and it is impossible to determine Kapp (Figure 3–8). There are other graphing techniques that can solve this problem such as an Eadie-Hofstee plot (Figure 3–9),

(Whikehart, Montgomery, Hafer, 1987).

Figure 3–8

The inability of a Lineweaver-Burk plot to give accurate information about the apparent K of an allosteric enzyme.

The degree of curvature and any intersection with the x-axis cannot be determined.

Enzymes • 63

Figure 3–10

Lineweaver-Burk plots for three inhibition mechanisms found in MichaelisMenten enzymes. In competitive inhibition, the apparent K is affected by competition of the inhibitor for the active site. In noncompetitive inhibition, the velocity of the enzyme is affected by the proximity of the bound inhibitor to the active site. In uncompetitive inhibition, both the apparent K (for one of the substrates) and the velocity of the enzyme are affected by the binding position of the inhibitor.

Figure 3–11

The binding positions and effects on enzyme catalysis produced by the three kinds of inhibition of Michaelis-Menten enzymes. In competitive inhibition, the substrate is blocked from entering the active site. In noncompetitive inhibition, the substrate enters the active site, but cannot easily be converted to product. In uncompetitive inhibition, a second necessary substrate is blocked from entering the active site.

64 • Biochemistry of the Eye

Figure 3–12

Plot of v vs. [S] when an inhibitor binds to an allosteric enzyme. The curve is shifted to the right causing both a decrease in velocity and a higher apparent K. The enzyme is in its T-form in the presence of an inhibitor.

catalyzed reactions having a specific purpose such as the breakdown of sugar molecules). In a series of such reactions, the end product of the series may actually act as an inhibitor for the first reaction in that series (feedback inhibition). The hydrogen ion content (pH) also influences enzyme reaction rates both positively and negatively. Intracellular pH varies from one cell type to another and is not necessarily equivalent to the extracellular or physiological pH of 7.4. This is important since most enzymes function intracellularly to control normal cell operation. Some enzymes act at cell membranes to facilitate transport and a number also catalyze reactions extracellularly. Tear film lysozyme is an example of an extracellular enzyme associated with the eye.

Lysozyme

Lysozyme is an enzyme of the precorneal tear film that is instrumental in destroying certain kinds of bacteria (namely, those which are positively stained with a crystal violet-iodine complex for peptidoglycans known as Gram stain). These Gram-positive bacteria possess an outer coat of a peptideglycan (sugar) polymer (or peptidoglycan), which, in Gramnegative bacteria, is only transiently stained since those bacteria are covered up by a second, outer lipid membrane. Lysozyme is able to hydrolyze or break up the glycan (sugar polymer) components of the peptidoglycan of Gram-positive bacteria as shown in Figure 3–13. The enzyme was initially described in 1922 by Alexander Fleming, a British bacteriologist (Stryer, 1988). He first found it in nasal mucous, but later discovered that tears are a rich source of the enzyme. The concentration has been estimated at 1.3 mg per ml of tears, which are unstimulated by external or internal sources such as onion odor or emotional stress (Sen and Sarin, 1980). Specifically, the enzyme breaks the β1→4 glycosidic bond of the oxygen bridge between the repeating glycan units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) as indicated in Figure 3–14. Lysozyme itself is a globular protein with a

Enzymes • 65

66 • Biochemistry of the Eye

Figure 3–15

Outline diagram of a lysozyme molecule. The enzyme is a globular polypeptide that has a groove (darker area near the top of the enzyme) into which the carbohydrate units of the peptidoglycans of bacteria can fit. Hydrolysis (or rupture) of the peptidoglycan units occurs there.

molecular weight of about 14,000. A portion of the bacterial peptidoglycan is able to fit in a groove on the outer face of the enzyme that contains the active site (Figure 3–15). This is an enzyme in which the detailed mechanism for hydrolysis is known and can be described in three stages. Figure 3–16 shows the mechanism in which the disaccharide unit is hydrolyzed. The active site contains two amino acid components (Glu and Asp) whose carboxylate groups participate in the hydrolysis. Initially, a proton (H+ from the Glu) breaks the bond by binding to the oxygen between the two sugar rings leaving an unbound, positively charged carbonium ion (carbon #4) in the right hand sugar ring. This carbonium ion is temporarily stabilized by the negative charge on Asp located above it (see Figure 3–16 A and B). Then, a nearby water molecule ionizes and donates its proton to the negatively charged Glu while the hydroxy group (–OH) binds to the carbonium ion and the reaction is complete (see Figure 3–16 B and C). At completion, the original forms of the enzyme are regenerated and the hydrolyzed (split) chains of the peptidoglycan leave the active site of the enzyme.

Once the peptidoglycan cover is split open (hydrolyzed) by lysozyme, the bacterium is no longer able to contain its high, internal osmotic pressure with its plasma membrane alone and it bursts open. Other protein components of tear film have also been implicated in bacteriocidal action (Selsted, Martinez, 1982), but none of them are as efficient as lysozyme.

In addition to its bacteriocidal activity, lysozyme also serves as an important analytical indicator of tear dysfunction. Measurement of lysosomal activity reflects the productivity of the main and accessory lacrimal glands (Gillette, Greiner, Allansmith, 1981) as well as the status of aqueous deficiency of the tear film (Van Bijsterveld, 1974; Van Bijsterveld, Westers, 1980). In the application of a Micrococcus agar diffusion assay (also known as a Schirmer Lysoplate Assay), for example, tear film is collected on filter paper discs and placed on a dish with agar containing 5 × 107 organisms of Micrococcus lysodeiticus. The lysozyme in the tear sample is allowed to hydrolyze the peptidoglycans and destroy

Enzymes • 67

Figure 3–16

Molecular mechanism of lysozyme catalysis at the active site. (A) A proton is donated by an uncharged Glu residue breaking the glycosidic bond while forming a hydroxyl group in the left hand sugar and a charged carbonium ion (also known as a carbocation) at carbon 4 of the right hand sugar. A negatively charged Asp on the enzyme stabilizes the carbonium ion. (B) A nearby water molecule ionizes to add a hydroxyl group to the carbonium ion and a proton back to the Glu on the enzyme. (C) The two fragments of the peptidoglycan are released from the active site.

68 • Biochemistry of the Eye

Figure 3–17

The Micrococcus agar diffusion assay.

The tear sample containing lysozyme clears an area (zone of lysis) in an agar base (a gelatin-like product of seaweed) containing a standard amount of the bacterium: Micrococcus lysodeiticus. The amount of lysozyme is proportional to the area cleared.

the bacteria for 24 hours at 37°C. Then the diameter of the clear area of agar (i.e., destroyed bacteria) surrounding the tear sample is measured (Figure 3–17). This cleared diameter may be converted to units of enzyme activity or simply interpreted either as normal or indicative of tear dysfunction. More recently, Klaeger and coworkers have developed a more reliable and efficient assay in which enzyme activity is measured from collected tears with the substrate: p-nitrophenyl penta-N-acetyl β-chitopentaoside. This substrate releases the colored product: p-nitrophenol upon enzyme hydrolysis and tear sample lysozyme activity can be analyzed within one hour.

Na, K-ATPase

Sodium, potassium-stimulated adenosine triphosphatase is an enzyme located in the plasma membranes of a wide variety of cells, but in ocular tissues it has two special functions: (1) control of corneal hydration and;

(2) the production of aqueous fluid. The enzyme is membrane-bound, which is to say that it is an integral protein that spans the width of cell plasma membranes. Its minimal quaternary structure is strongly postulated to consist of four polypeptide chains: two α and two β chains. This is shown in Figure 3–18. The α chains are the actual catalytic molecules for which the substrate is the high energy compound: ATP (adenosine triphosphate, see Chapter 4). The catalytic reaction is:

Na, K ATPase→

−

ATP ADP + P

i

Beyond this, the catalyzed reaction is energetically coupled to an ion transport process. Inorganic phosphate (Pi) becomes bound to one of the α-subunits and in the process supplies the energy necessary to transport three sodium ions out of a cell and two potassium ions inward. The exact detailed mechanism remains elusive. However, it is postulated that this may take place either by a conformational shuttle within the α subunits or by the existence of pores in the subunits through which the ions are pumped.