ABI 102 Exam 3

membrane potential

the difference in electric potential across a biological membrane

how can passive diffusion of K lead to development of negative membrane potential?

K leaks out of the cell, taking its positive charge with it and leaving the inside more negative

non mediated transport

simple diffusion, substance diffuses in direction that eliminates its gradient, eg: steroids and O2

mediated transport

passive mediated and active transport, requires specific carrier proteins

passive-mediated transport (facilitated diffusion)

specific molecule flows from high to low

active transport

specific molecule is transported from low to high, against the concentration gradient, endergonic, coupling required

ionophores

organic molecules that increase the permeability of membranes to ions, carrier and channel-forming

carrier ionophores

bind selected ions, diffuse them through membrane, release ion to other side, and return to original side, must be soluble in nonpolar solvents

channel-forming ionophores

transmembrane channels through which their selected ions can diffuse

porins

a passive-mediated transport channel that is a B-barrel structure through which solutes selectively pass through the channel

ion specific channels

allow rapid passage of ions like K+, Na+, Cl-, essential for maintaining osmotic balance, signal transduction, effecting changes in membrane potential that are responsible for neurotransmission

K+ channels

allow K+ ions to passively diffuse, over 10,000x more permeable to K+ than Na+, some are consecutively open to keep the negative membrane potential

KcsA structure

8 helices, 4 subunits each with two helices, negative charge at base to repel negative ions, until reaching selectivity filter, a K+ ion is surrounded by water molecules which help the ion through the pore rapidly, allows for ease of movement because of the hydration shell with 8 H2O molecules, within the filter, O molecules form H2O and protein contribute to K+ selectivity, repulsion of regularly spaces K+ ions

gated ion channels

opening and closing of ion channels, response from various simuli

mechanosensitive channels

open in response to deformation in the lipid bilayer (touch, sound, pressre)

ligand-gated channels

open in response to an extracellular chemical stimulus (neurotransmitter)

signal-gated channels

opens on intracellularly binding Ca2+ ion or some other signaling molecule

voltage-gated channels

open in response to a change in membrane potential (nerve impulse)

depolarization

local increase in membrane potential where membrane becomes less negative

repolarization

process of returning to resting membrane potential after depolarization, ions flow out of the cell

hyperpolarization

increase in membrane potential where membrane becomes more negative than resting potential.

aquaporin

a protein pore that conducts water through biological membranes

AQP1

a type of aquaporin that facilitates water transport in various tissues, critical for maintaining fluid balance. six transmembrane a helices arranged like an hourglass and two shorter helices lying within the bilayer

• pore lined with hydrophobic groups

• Arg and His residue regions and backbone carbonyls “lure” H-bonding H2O molecules

• conserved Asn within center and thereafter so NH2 groups can H-bond with H2O to reorient molecule so one molecule leaves at a time

gap junctions

made up of connexions, a single gap junction consists of two hexagonal rings of connexins called connexons. Small molecules and ions can pass through, join discrete regions of neighboring cells’ plasma membranes, each connexon contain 6 connexin subunits

GLUT1 (Erthrocyte Glucose Transporter)

a type of glucose transporter that facilitates the passive transport of glucose across the plasma membranes of mammalian cells, critical for energy metabolism.

Uniport

movement of a single molecule at a time

symport

simultaneously transports two different molecules in the same direction

antiport

simultaneously transports two different molecules in opposite directions

P-type ATPases

undergo phosphorylation as they transport cations like Na+, K+, and Ca2+ across the membrane

A-type ATPases

transport anions across membranes

ABC transporters

have ATP-binding cassette and transport a wide variety of substances, including ions, small metabolites, and drug molecules

primary active transport

uses chemical energy (from ATP)

secondary active transport

uses electrochemical potential

(Na+-K+)-ATPase

Transports ions in opposite directions, transmembrane protein that depends highly upon the phosphorylation of an Asn

• happens clockwise because of reaction coupling

• exergonic steps: step 2, step 5, ATP binding in step 6

• endergonic steps: step 3,4, and K+ release in step 6

(Na+-K+)-ATPase E1 state

picks up ATP, Na+ forms high energy aspartyl phosphate intermediate and makes E1 a high energy state, allowing Na+ to be released into the cytoplasm.

(Na+-K+)-ATPase E2 state

sodium is outside of the cell, phosphate still attached and signals to pick up K+ on outside of cell, phosphate get hydrolyzed and removed, ATP interacts on the inside of the cell and pulls the K+ inside the cell

Ca2+- ATPase

increase in cytosolic [Ca2+] triggers cell responses like muscle contraction, neurotransmitter release, and glycogen breakdown, pumps our 2Ca2+ and brings in 2-3 H+ at the expense of ATP hydrolysis

glucose transport in intestinal epithelium

cells of the intestinal take up glucose by Na+-dependent symport. one glucose and two Na+ enter through the symport, glucose enters uphill of its gradient (more glucose in cell than lumen).

How is the Na+glucose symport effective

Na+ is leaving the cell on the basolateral side of the cell as a part of Na+-K+-ATPase active transport. 3 Na+ leave the cell by the ATPase and the gradient is maintained, glucose is transported into the cell and passed to the capillaries via passive-mediated uniport.

general properties of enzymes

higher reaction rates, milder reaction conditions, greater reaction specificity, capacity for regulation

how do enzymes act on specific substrates

substrates bind to enzymes w noncovalent character similar to protein conformation: van der waals, electrostatic, h-bonding, hydrophobic interactions, shape, charge, induced fit

enzyme stereochemistry

chiral, bind with stereospecificity to one stereoisomer of a substrate (asymmetric active sites),

coenzymes

organic molecules required for enzyme catalysis, bind to the enzyme’s active site and participate in catalysis but are not considered substrates of the reaction

cosubstrates

type of coenzyme, only transiently associated with the enzyme, not attached and sometimes recycled

prosthetic groups

type of coenzyme, organic and inorganic with no amino acids, bound tightly proteins (maybe covalently), always bound to enzyme

what happens when you add a catalyst to activation energy

catalyst provide a reaction pathway with a transition state whose free energy is lower than that in the uncatalyzed reaction, thus lowering the activation energy required for the reaction to proceed. the likelihood a reaction occurring in one direction or the other depends upon the free energy difference between the reactants and the products

rate enhancement

ratio of the rates of catalyzed and uncatalyzed reactions

general acid catalysis

a process in which proton transfer from an acid lowers the free energy of a reaction’s transition state

general base catalysis

a process in which a proton’s abstraction by a base lowers the free energy of a reaction’s transition state

concerted acid-base catalysis

both processes are working simultaneously on the reaction

• side chains of residues can act as acid-base catalysts

• enzyme can have several of these residues in a site

• enzymes sensitive to pH (side chain pKas)

covalent catalysis

accelerates reaction rates through the enzyme forms covalent bond with substrate by a nucleophilic attack (substrate is electrophilic group), thus stabilizing the transition state. the covalent bond releases, driving the reaction forward.

metal ion catalysis

1/3 enzymes require metal ions for catalytic activity, metal ion can acy like a proton to neutralize negative charge, yet are often more effective becasue they can be present in high amounts without affecting pH and can have charges over +1. metal ions participate in the catalytic process in three ways:

1. binding substrates to orient them properly for rnx

2. mediating oxidation-reduction reactions through reversible changes in the metal ions oxidation state

3. by electrostatically stabilizing or shielding negative charges

catalysis proximity and orientation effects

four common mechanisms:

1. bring substrates in contact w enzyme’s catalytic groups, and to other substrates

2. bind substrates to set up proper orientation of substrate for rxn

3. charged groups on enzyme can stabilize the transition state of the reaction (electrostatic catalysis) (charges guide substrates toward binding site)

4. freeze out the relative translation and rotational motions of substrates and catalytic groups

how do enzymes catalyze by preferentially binding the transition state

the transition state is the high energy, unstable midpoint of a reaction where bonds are in the process of breaking and forming, releasing less free energy difference between an enzyme substrate complex and an enzyme transition state complex than the free energy difference between the substrate and uncatalyzed reaction

transition state analog

stable molecules that geometrically and electronically resemble the transition state, serve as potent enzyme inhibitors if an enzyme preferentially binds its transition state

enzyme kinetics

study of enzymatic reaction rates: E + S <—>ES—> P + E

what happens if [S] is high enough to entirely convert the enzyme to the ES form?

the second step of the reaction becomes rate-limiting and the overall rxn rate becomes insensitive to further increases in substrate.

rate constant (k)

proportionality constant in the equation that expresses the relationship between the rate of a chemical reaction and the concentrations of the reacting substances

production of ES from both directions

dES/dt= (k1[E][S]) - (k-1[ES]) + (k2[ES])

Ks when E has good affinity for S

Ks is small Ks=k-1/k1=[e][s]/[es]

Ks when E has less affinity for S

Ks is larger, Ks=k-1/k1=[e][s]/[es]

ES steady state

substrate will always become product at a constant rate, there’s a lot of substrate initially

Michaelis constant

Km=(k-1+k2)/(k1)

Km is the [S] at which the reaction rate is at half-maximum

It is an important parameter that reflects the affinity of the enzyme for the substrate; a lower Km indicates higher affinity.

Michaelis-Menten Kinetics equation

V0= (Vmax[S])/([S]+Km), graphed as a parabola

Ks

dissociation constant of Michaelis complex

As Ks decreases, enzyme’s affinity for substrate increases

As Km decreases, enzyme’s affinity for substrate is stronger

Kcat

Catalytic constant, turnover number, number of reaction processes that each enzyme catalyzes per unit time, same as how fast you get product (K2)

inhibitors

substances that reduce an enzyme’s activity, influence binding, enzymes speed, can be reversble, ‘inactivator’ if irreversible, can resemble the substrate structurally

competitive inhibition

substance that directly competes with a substrate for an enzyme’s binding site, may resemble the substrate or just not react, competitive inhibitors reduce the concentration of free enzyme available for substrate binding

uncompetitive inhibition

binds directly to the enzyme substrate complex but not to the free enzyme, distorts the active site thereby rendering the enzyme catalytically inactive [ESI], adding more substrate does not reverse the effect of an uncompetitive inhibitor because substrate binding does not interfere with uncompetitive inhibitor binding.

noncompetitive/mixed inhibition

binds to the enzyme sites that participate in both substrate binding and catalysis