Ocular Biochem: Enzymes

Enzymes

-proteins that optimize the rates of biochemical reactions in cellular tissues

-involved in several biochemical reactions

What are the roles of enzymes in the eye?

-visual transduction

-generation of IOP

-maintenance of clear cornea

-destruction of bacteria in tear film

-development of lens fibers and several other functions

How do enzymes increase the rate of a reaction?

-act by lowering the activation energy to convert a reactant into a product

-at least one intermediate is formed prior to product formation

-comparatively small amounts of energy is required to form the product in presence of a catalyst than without a catalyst

-reactions are usually reversible, a large amount of substrate (A) will drive the reaction to form product (C) and vice versa

Molecular Events of Enzyme Catalyzed Reaction

-in this reaction, a substrate is bound to a region of the enzyme known as the active site

-small changes such as conformational change occur to the enzyme as a part of the catalytic event

-the enzyme is always returned to its original form at the end of each reaction

Active Site

-a cleft on the outside or a small cavern on the inside of the structure of the catalytic protein

-suitable for holding the substrate in a favorable orientation

-this facilitates rapid conversion first to an intermediate and then to a product

What are the different classifications of enzymes based on the kinds of reactions?

-oxidation-reduction

-transfer of molecular groups

-hydrolytic cleavage

-double-bond alteration

-isomerization

-condensation

another way to classify enzymes is on a kinetic basis

Michaelis-Menten Enzymes

-have kinetic functional properties

-reaction kinetics are derived from [E] + [S] <(k1)--(k2)> [ES] --(k3)> [P]

[E] = molar concentration of enzyme

[S] = molar concentration of substrate

[ES] = molar concentration of enzyme-substrate complex

[P] = molar concentration of product

k1, k2, and k3 are rates for each conversion to and from [ES]

k3 = aka k(cat) (turnover number) when [S] is high and v = Vmax

What occurs when there are changes in concentration in enzyme catalyzed reactions?

-enzyme becomes saturated very early in time with substrate due to formation of enzyme-substrate complex

-remains at a fairly steady level

-change in [ES] may be described at d[ES]/dt = 0

-since the change in [ES] is very small or nearly zero, d[ES]/dt can also be described in terms that cause both its formation k1[E][S] and its dissolution k2[ES] + k3[ES]

Michaelis-Menten Equation

v = Vmax[S]/[S]+Km

Vmax = the maximum velocity of the enzyme

Km = (k2 + k3)/k1 = Michaelis-Menten constant

Michaelis constant is actually a dissociation constant for the [ES] complex in which [ES] --> [E] + [S]

What does the Michaelis-Menten equation mathematically define?

-maximum velocity (Vmax)

-dissociation constant (Km) of an enzyme

What are the terms in the Michaelis-Menten equation useful for?

-measuring the rate properties of an enzyme (how fast they catalyze reactions)

-comparing the relative affinity (stickiness) of different substrates for the same of different enzymes

What is the signifiance of the Michaelis-Menten constant (Km)?

-lower the Km value, the greater will be the affinity of substrate and enzyme provided that k3 is sufficiently small

-if this is not the case, then the term Km or Kapparent is used

-this term also indicates the presence of other substances or conditions which may modulate enzyme's activity

When is Km equal to the substrate concentration [S]?

when v is half Vmax

Where does Km lie for most enzymes?

between 1x10^(-1) and 10^(-7)

Why are Vmax and Km of an enzyme not usually measured by constructing a graph?

due to difficulty in obtaining the value of Vmax as it reaches an asymptotic limit

Lineweaver-Burk Plot

-it is a double - reciprocal plot and another approaching of finding Vmax and Km

-plot is constructed by using the reciprocal of the Michaelis-Menten equation

-this equation helps to obtain a straight-line graph having two intercepts (x and y), which give the reciprocal values for Km and Vmax respectively

What is an example of a Michaelis-Menten enzyme?

aldose reductase - involved in formation of diabetic cataracts

What is the application of the lineweaver-burk plot?

-enzyme velocities are measured as the concentration of substrate consumed or product formed per unit time at stated conditions of temperature

-x-intercept is a negative value

Allosteric Enzymes

-allosteric = other site

-are proteins with quaternary structures, each chain having its own active site

-when only one or a few active sites are occupied, the affinity of enzyme and substrate is low

-in this situation, the velocity of the reaction is also low

What are the kinetics of allosteric enzymes influenced by?

-influenced by substances bound to the enzyme at locations other than active site

-also influenced by binding of activator substances that occupy sites away from the active site

What occurs to allosteric enzymes as the number of active sites occupied increases?

-overall conformational change in the enzyme occurs

-becomes more favorable to increased binding of the substrate to the remaining active sites which increases the velocity of the enzyme

-changes conformation from a T-form (tense) to an R-form (relaxed)

How does the binding of an activator affect an allosteric enzyme?

-facilitate changing from T to R form at lower substrate concentrations

-this is a biological way of jump-starting an enzyme to high velocities at lower substrate concentrations

What occurs when an activator is bound to allosteric enzymes?

-apparent K is decreased

-velocity is increased

-with allosteric enzymes, Km becomes Kapparent since the K value is dependent on activators as well

What is an example of an allosteric enzyme?

sodium potassium ATPase - an enzyme important for cornea and ciliary body

When does the lineweaver-burk plot become non-linear?

-becomes non-linear with allosteric enzymes and it is impossible to determine Kapp

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

Eadie-Hofstee Plot

-used to determine Kapp of allosteric enzymes

-initial velocity is plotted against the initial velocity divided by the activator concentration of the enzyme

-sodium activation for Na, K-ATPase present in fresh tissue and tissue cultures of BCEC can be determined

-value of v/[S] or v/[A] is refined by using a hill coefficient

-values of Kapp in mM for Na activation are at the Y intercepts of the plot

Where are inhibitors that influence the rate of enzymes located?

-present within tissues OR

-artificially introduced into tissues

What enzyme is used for a treatment approach for glaucoma?

involves inhibition of enzyme acetylcholinesterase, an enzyme that lyses acetylcholine in the ANS e.g. neostigmine

What type of mechanisms can Michaelis-Menten enzymes be inhibited by?

-competitive

-noncompetitive

-uncompetitive

How does enzyme inhibition affect lineweaver-burk plots?

indicate how the apparent Km and Vmax may be influenced by the mechanism of inhibition

What is the role of 1+[I]/Ki in lineweaver-burk plots when there is enzyme inhibition?

-becomes a factor in all three forms of inhibition

-used to determine the concentration of the inhibitor and its affinity for the enzyme

-[I] = molar concentration of the inhibitor

-Ki = a measure of the affinity of the inhibitor for the enzyme

Competitive Inhibition

inhibitor replaces or competes with the substrate for active site

Non-Competitive Inhibition

-inhibitor binds to a site close to the active site

-inhibitor prevents catalytic action on the substrate even though it may bind to the enzyme

Uncompetitive Inhibition

-two substrates are usually required for catalytic action

-inhibitor binds to an area close to the active site after the first substrate binds there

-inhibitor then prevents the second substrate from binding

-the latter mechanism occurs with enzyme aldose reducatase which is involved in formation of diabetic cataract and galactosemia

What is the inhibition of allosteric enzymes like?

-inhibited by substances binding to allosteric sites

-this results in a more pronounced T-form which makes it more difficult for the substrate to bind to the enzyme

-inhibition also serves to slow down the rates of metabolic reactions

-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)

What is the influence of pH on the enzyme reaction rate?

-hydrogen ion content (pH) influences enzyme reaction rates both positively and negatively

-intracellular pH varies from one cell type to another and is not equivalent to the physiological pH of 7.4

Extracellular Enzymes

-some enzymes act extracellularly at cell membranes to facilitate transport and extracellular reactions

-tear film lysozyme is an example of extracellular enzyme associated with the eye

Lysozyme

-enzyme of the precorneal tear film

-helps in destroying gram-positive bacteria

-globular protein with a MW of 14,000

-concentration in tears = 1.3 mg/ml

How do lysozymes destroy gram-positive bacteria?

-gram-positive bacteria possess an outer coat of a peptidoglycan

-gram-negative bacteria have a second, outer lipid membrane in addition to peptidoglycan

-lysozyme hydrolyzes the glycan (sugar polymer) components of the peptidoglycan of gram-positive bacteria

What is the process of lysozymes destroying gram-positive bacteria?

-it specifically breaks the β1 --> 4 glycosidic bond of the oxygen bridge between the repeating glycan units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)

-a portion of the bacterial peptidoglycan fits in a groove on the outer surface of the enzyme that contains the active site

Mechanism of Action of Lysozyme

-active site of lysozyme contains two amino acid components (glutamic acid and aspartic acid) whose carboxylate groups participate in the hydrolysis

-initially, a proton breaks the bond by binding to the oxygen between the two sugar rings

-this creates 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 aspartic acid located above it

-then a nearby water molecule ionizes

-proton is donated to the negatively charged Glu while the -OH group binds to the carbonium ion and the reaction is complete

What occurs after the completion of the reaction in the mechanism of action of lysozymes?

-at completion of reaction, the original forms of the enzyme are regenerated

-hydrolyzed (split) chains of the peptidoglycan leave the active site of the enzyme

-following the splitting of peptidoglycan cover by lysozyme, the bacteria bursts open

-bacteria can no longer contain its high osmotic pressure with plasma membrane alone

What are the applications of measurement of lysosomal acitivity?

-to assess productivity of the main and accessory lacrimal glands

-assess status of aqueous deficiency

Schirmer Lysoplate Assay

-tear film is collected on filter paper discs

-discs are placed on dish containing 5x10^7 organisms of Micrococcus lysodeiticus

-lysozyme in the tear sample is allowed to hydrolyze the peptidoglycans and destroy the bacteria for 24 h at 37 c

-after 24 h, the diameter of the clear area of agar (destroyed bacteria) surrounding the tear sample is measured

-cleared diameter may be converted to units of enzyme activity or interpreted as normal or indicative of tear dysfunction

What are some other techniques for lysozyme activity?

-recently, activity is measured using the substrate p-nitropheyl penta-N-acetyl β-chitopentaoside

-this substrate releases the colored product p-nitrophenol upon enzyme hydrolysis

-tear lysozyme activity can be analyzed within one hour

Where is Na, K-ATPase located?

in the plasma membranes of a wide variety of cells

What are the two special functions of Na, K ATPase in ocular tissues?

-control of corneal hydration

-production of aqueous fluid

Na, K-ATPase

-a membrane bound integral protein that spans the width of cell plasma membranes

-quaternary structure consists of four polypeptide chains two α and two β chains

-α chains are the actual catalytic molecules for which the substrate is the high energy compound: ATP

-beyond this, the catalytic reaction is energetically coupled to an ion transport process

-inorganic phosphate (Pi) becomes bound to one of the α-subunits

-Pi supplies the energy necessary to transport 3 sodium ions out of a cell and 2 potassium ions inward

How can the transport of three sodium ions out of a cell and two potassium ions inward take place?

-by a conformational shuttle within the α subunits OR

-by the existence of pores in the subunits through which the ions are pumped

What is the role of Na, K-ATPase in the eye?

-helps in removal of excess water from stroma by pumping sodium ions into the channel where they flow into the anterior chamber following the path of least resistance (diffusion)

-water follows sodium ions as an osmotic function

-in the absence of pumping mechanism, Na ions and water would continuously enter the corneal stroma, causing it to swell and become opaque

How does Na, K-ATPase indirectly generate IOP in the eye?

-operates in the NPE cells of the ciliary body by a similar mechanism

-a surplus of Na ions are pumped into the posterior aqueous chamber by Na, K-ATPase

-this causes water to flow into the chamber osmotically

-the enzyme indirectly generates an intraocular pressure

Lactate Dehydrogenase (LDH)

-a protein tetramer with a MW of 140,000

-operates at the metabolic junction of aerobic and anaerobic metabolism

-found in the cytoplasm of all eukaryotic cells

What are the two substrates for LDH?

-pyruvate is the metabolic substrate for the reaction and is formed from glucose, fructose, and other carbohydrates

-Coenzyme NADH is the second substrate that provides electrons for the reduction of pyruvate to lactate

What are the three polypeptides of LDH?

-H = heart

-M = muscle

-K = subunit found in cancer cells

various combinations of these polypeptides customize the activity of LDH

K4 or LDH4 type

-found in photoreceptors, cancer cells

-characteristic of cells with very high metabolic rates

Isozymes

different forms (subunits) of the same enzyme

What do the kinetic properties of each enzyme for LDH determine?

-the relative percentage of aerobic and anaerobic pathways to be used by the cells

-whether the cell will obtain quick energy (anaerobic) or fuel efficient energy (aerobic)

H4LDH Enzyme

lactate formation is impeded and pyruvate formation is favored

M4LDH

permits the reaction to proceed quality in either direction

K form of LDH

-one whose catalytic rate increases with the partial pressure of oxygen

-Keq approaches 1 as the pO2 increases

What is the role of LDH in the corneal epithelium?

-corneal epithelial cells normally obtain oxygen from the precorneal tear film

-corneal epithelial cells need to survive during low partial pressures of oxygen which can occur during lid closure and contact lens wear

-under such conditions, epithelial cells must shunt their carbohydrate metabolism to lactate production via LDH

-this mechanism can successfully support epithelial cells at low oxygen partial pressures (15-20 mm Hg)

What are the percentages of different isozyme forms of LDH in the human cornea?

-H2M2: 25%

-HM3: 65%

-M4: 10%

combination of the above isoforms would be favorable for lactate production under conditions of oxygen starvation

What is the role of LDH in the lens?

-in whole lens, the dominant population of cells are lens fiber cells whose metabolic rate is low compared to most cells

-lens fiber cells make use of very little oxygen for metabolism

-in lens, only HM3 and M4 LDH isozymes are found

What is the role of LDH in the retina?

-LDHk opens the pathway to lactate when no further pyruvate can be driven through aerobic pathway

-this helps in rapid metabolism of glucose since the energy demands in photoreceptors is very high

-LDH(k)M4

LDH(k)M4

-modified form of LDH(k) and differs in two ways

-phosphate is bound on tyrosine residue #238 near the active site causing a conformational change

-the enzyme is bound to other proteins

-either one or both changes makes it sensitive to oxygen

Warburg Effect

phenomenon of high lactate production coupled with high glucose and oxygen consumption

production of lactate in retina is higher than in any other aerobic tissue

Aldose Reductase

-belongs to a family of aldo-keto reductases and exists in globular form

-MW range: 28,000 to 45,000; consists of a single polypeptide chain

What is the activity of aldose reductase associated with?

-associated with cataract formation in diabetics and in patients having galactosemia

-it catalyzes the conversion of glucose to sorbitol and galactose to galactitol

What are sorbitol and galactitol referred to as?

polyols

Polyols

-induce an osmotic imbalance in lens fiber cells

-cause them to swell and eventually burst since they cannot exit the cell

What is the role of aldose reductase in diabetes?

-aldose reductase is inactive in the lens until the concentration of glucose or galactose rises to cause its activation

-significant activation of aldose reductase occurs when blood sugar levels approaches 20mM for sustained periods

What are the applications of an inhibitor for aldose reductase?

-anticataractogenic agent

-prevention of diabetic retinopathy

What are the different sites on aldose reductase?

-there is an inhibitor site on the enzyme that is lipophilic

-this site is capable of associating with a variety of fat soluble substances that may act as inhibitors

-the binding sites for sugar, NADPH, and an inhibitor is in a separate, but nearby location

What are the three inhibitors tested for aldose reductase?

-quercetin: too weak inhibitor

-sorbinil: causes a high degree of hypersensitivity reactions

-tolrestat: recently tested compound

no inhibitor has been able to meet FDA standards

What type of inhibition do the tested inhibitors of aldose reductase use?

either uncompetitive or noncompetitive inhibition - mechanism depends on the inhibitor used

Matrix Metalloproteinases

-are protein hydrolases

-involved in breakdown of ECM or noncellular tissue architecture

-control functions such as growth and development, tissue maintenance, tissue reformation and deterioration

-requires zinc at the catalytic site to carry out its lytic activity

How many members of the matrix metalloproteinases family have been identified?

17 members of the family

Gelatinases

a matrix metalloproteinase specific for types IV and V collagens

Stromolysins

a matrix metalloproteinase specific for noncollagenous ECM proteins

What is the role of matrix metalloproteinases in the eye?

-remodeling of eye's axial length with the development of myopia

-destruction of corneal collagen following alkali and other chemical burns

-hydrolyze a variety of ECM proteins found in the eye

What is the mode of action of matrix metalloproteinases?

-collagenases 1 and 2, stromelysin 1, matrilysin, and gelatinases A and B hydrolyze the Gly-ile bond of the following sequence equally well:

Gly-Pro-Gln-Gly^-Ile-Ala-Gly-Gln

-minor variations such as substituion of valine for glycine on amino acid #3 will cause a considerable variation in specificity and activity of individual matrix metalloproteinases

What are the components of matrix metalloproteinases?

-signal peptide

-propeptide

-furin cleavable site

-catalytic domain

-hinge region

-homopexin domain

-transmembrane domain and cytoplasmic tail

Signal Peptide in Matrix Metalloproteinases

directs the cell to move the newly synthesized enzyme into ER for transport out of the cell

Propeptide in Matrix Metalloproteinases

-maintains the enzyme in its inactive form until needed by inhibiting zinc

-activation of the enzyme is dependent upon changes or chemical signals made to the propeptide region

Furin Cleavable Site in Matrix Metalloproteinases

-brings about activation of the enzyme

-furin is a golgi associated proteinase that cleaves the propeptide region to cause activation

Catalytic Domain in Matrix Metalloproteinases

-contains zinc ion and also binds to calcium ions

-carries out the catalytic activity of the enzyme

Hinge Region in Matrix Metalloproteinases

connects the catalytic domain with the homopexin domain

Homopexin Domain in Matrix Metalloproteinases

-substrate specificity binding

-binding to a natural inhibitor, a TIMP (tissue inhibitor of metalloproteinase)

Transmembrane Domain and Cytoplasmic Tail in Matrix Metalloproteinases

-used to physically extend the enzyme into cells that express matrix metalloproteinases

-serve as an additional molecular mechanism for activation of the enzyme

What is the role of matrix metalloproteinase 2 (MMP2, Gelatinase A) in myopia?

-in myopia, the ocular globe is lengthened along its anterior-posterior axis

-this is due to remodeling or reformation of ECM proteins in the posterior sclera

-mRNA used to synthesize gelatinase A increased by 125% over its control tissue

-mRNA used to synthesize TIMP-2 decreases by 75% of its control tissue

-when form deprivation was discontinued for 24 hours, mRNA for gelatinase A decreased 50% of control and mRNA for TIMP-2 increased by 12%

-results suggest that controlled gelatinase A activity plays a role in the process of lengthening the axis of the globe when myopia is induced

-this occurs as a result of partial degradation of scleral proteins, and new proteins formed as a result of this process establish a new scleral length

What is the role of matrix metalloproteinases in the cornea when there is an acute alkali burn?

-damaged proteins are removed from the cornea by the action of MMPs

-process weakens the cornea leading to its perforation and loss of the eye

What is the role of matrix metalloproteinases inhibitors in the cornea when there is an alkali burn?

-alkali burned corneas when exposed to B mercaptomethyl-Leu-Phe-Ala (a SIMP, synthetic inhibitor of metalloproteinase) burned tissue maintained at a clinical score of ~1 for 15 days (1 - superficial ulcer at a depth of no more than one third of the anterior tissue)

-tissues not exposed to the SIMP achieved a score of 3 or worse after 4 days

-ulcer formation was equally inhibited upon exposure to TIMP inhibitor (a naturally secreted polypeptide with the MMP)