Biochem exam 2

Mass Spectrometer

converts molecules to ions so they can be moved about and manipulated by external electric and magnetic field

The Ion Source

small sample is ionized, usually to cations by loss of an electron

The Mass Analyzer

ions are sorted and separated according to their mass and charge

The Detector

separated ions are then measured and the results displayed on a chart

Protein Mass Spec

proteins are long polymers of amino acids, and are shortened to small peptides before MS

Mass Spectrometry

most common way to determine amino acid sequences by separating particles on the basis of mass-to-charge ratio

Electrospray Ionization (ESI-MS)

macromolecules sprayed from glass capillary as fine droplets without intense heat

Matrix Associated Laser Resorption Ionization Time of Flight (MALDI-TOF)

protein sample is mixed with a chemical matrix that includes a light absorbing substance excitable by a laser; laser pulse excites the chemical matrix; excitation of the matrix creates a micro plasma that transfers energy to the protein molecules that are then ionized and injected into gas phase

Tandem MS

two mass spectrometers that couple together; first is to isolate the peptide of interest, selected peptides enter the collision cell where it collides with helium, then the energy of the collision can cause a peptide bond to break resulting in a few smaller fragments; smaller fragments enter the second MS where their molecular masses are determined

Amino Acid Sequencing

Advantage: no prior knowledge or peptide necessary

Disadvantage: time consuming, dicomol of material needed

Peptide Mass Fingerprinting

used to identify unknown protein; can find the exact mass by MALDITOF

Homologs

same function different organisms

Orthologs

proteins from different species that have similar sequences and function

Paralogs

proteins within one species that have similar amino acid sequences

Protein Mutations

mutations at the genetic level are translated in different amino acids at the protein level

Prosthetic Group

coenzyme that is tightly bound to the enzyme; non-amino acid is important to the function of the protein

Glycoprotein

carbohydrate groups, covalent, immunoglobulins

Lipoprotein

lipids, covalent or noncovalent, blood lipoprotein complexes

Nucleoprotein

RNA/DNA, noncovalent, ribosomes, chromosomes

Metalloprotein

Metals, metal activated proteins, covalent or noncovalent, metabolic enzymes, kinases

Hemoprotein

heme group, covalent or noncovalent, hemoglobin

Flavoprotein

FAD, covalent or noncovalent, electron transfer enzymes

-omes

characterization/identification of all molecules or functions of particular class in a given ell of organisms; dynamic

-omics

field of study to classify the -ome in a particular field, proteins that are present at a certain snapshot of time; require large scale comprehensive analysis

Proteome

full genetic potential of a cell that is contained within its genome; more accurate refle4ction of what a cell is doing at any moment is found in this spot

Protein Rules

function depends on structure; structure depends on sequence and weak forces; number protein folding patterns is large; structures of globular proteins are marginally stable; marginal stability facilitates motion; motion enables function

Hydrogen Bonds Stabilize Protein Structure

amino acid backbone atoms can bond with other backbone atoms; amino acid side chain functional groups can bond, side chain bonds on protein surface mediate contact with water or with the surface of other polypeptide chains

Ionic bonds stabilize protein structure

electrostatic interactions can arise between positively and negatively charged amino acids

Van der waals interactions stabilize protein structure

instantaneous dipole-induced dipole interactions that arise because of fluctuations in the electron charge distributions of adjacent nonbonded atoms; contributes to the tightly packed interior of many globular proteins

hydrophobic interactions

nonpolar side chains prefer to cluster in a nonpolar environment rather than be exposed to water; clustering nonpolar residues in the core of the protein is entropically driven; hydrophobic interactions are the driving force behind protein folding

Secondary Structure

protein backbone structure is based on the amide plane; the planarity of the peptide bond means there are only two degrees of freedom per residue for peptide chain; each alpha carbon is the joining point for two planes defined by peptide bonds

Phi

angle about the Calpha- N bone

Psi

angle about the Calpha- C bond

Alpha-Helix

first proposed by Linus Pauling and Robert Corey in 1951, residues per turn 3.6, rise per residue 1.5, rise per turn (pitch) 5.4

Beta-Pleated Sheet

composed of beta strands, may be parallel (off set) or antiparallel (aligned), rise per residue antiparallel 3.47, rise per residue parallel 3.25

Fibrous Proteins

alpha-keratin, 311-314 alpha residue helical rod segments, helical rods consist of 7-residue re[eats, promotes association of helices to form coiled coils

Fibroin

silk fibers; formed from extensive beta-sheets alternating sequence; since residues of beta sheets extend alternately above and below the plane of a sheet, this places all glycine’s on one side and all alanine’s and serine’s on the other side

Collagen

triple helix, 3 intertwined polypeptide chains (300 nm length 1.4 nm diameter); unique amino acid composition includes hydroxyproline and hydroxy lysine

Membrane Proteins

can be peripheral or integral; can be made of alpha helices or beta sheets

Globular Proteins

most common, responsible for most of the functions in the cell; helices and sheets make up the core; protein core is predominantly nonpolar; highly polar N-H and C=O moieties of the peptide backbone must be neutralized in the hydrophobic core; very tightly packed with little empty spaces ; mostly hydrophobic residues force the interior to interact with each other

Globular Protein Core

helices and sheets in thec ore are typically uniform and conserved in sequence and structure

Globular Protein Surface

made of loops and tight turns that connect the helices and sheets of the core; surface elements can interact with small molecules or with other proteins many surface elements are the basis for enzyme-substrate interactions, cell signaling, and immune responses

Denaturation Leads to Loss of Protein Structure and Function

the cellular environment studied is suited to maintaining the weak forces that persevere protein structure and function; external stresses are heat, chemical treatment that can disrupt these forces in a process termed to denaturation

Denaturation Experiments Helped us Learn About Protein Folding

Ribonuclease A a small enzyme that cleaves chains of ribonucleic acid; 124 residues and 4 disulfide bonds; treated the proteins with urea (unfolds proteins) and mercaptoethanol (reduced disulfide bridges); complete loss of enzyme function

Anfinsen’s experiment

Demonstrated that proteins fold reversible byt we want to know more about the steps of protein folding; there must be some pathway to protein-folding otherwise a protein would never reach its final state in a reasonable timeframe

Thermodynamic Driving Force for Folding of Globular Proteins

if the free energy change is negative, the reaction is spontaneous; for folding of a globular protein, the free energy change must be negative if the folded state is more stable than unfolded state; the free energy change depends on changes in enthalpy and entropy for polar residues, nonpolar residues, and water

Protein Felxibility and Motion

proteins are marginally stable and not rigid, many noncovalent bonds can be interrupted, broken, rearranged; flexibility is important for protein function; ligand binding, enzyme catalysis, and enzyme regulation often require oscillation and fluctuation in protein structure

Atomic Fluctuations

vibrations of a few angstroms due to kinetic energy

Collective Motions

movement of a group of atoms over larger distances

Conformational Changes

movement of a whole section of the protein may be distances up to 1 nm

Quaternary Structure

assembly of multiple subunits; subunits of oligomeric proteins fold independently of each other and then interact; interacting surfaces must have complementary arrangements of polar and hydrophobic groups; subunits can be identical or non-identical

Oligomers

complexes are complexes composed of noncovalent assemblies of two or more monomer subunits

System (Thermodynamics)

the portion of the universe which we are concerned

Surroundings (Thermodynamics)

everything else

Thermodynamic systems

isolated system cannot exchange matter or energy; closed system can exchange energy; open system can exchange either or both

First Law of Thermodynamics

total energy of an isolated system is conserved; E2-E1=deltaE= q+w

Enthalpy

the heat content of a system; heat absorbed in constant pressure process; when heat is absorbed by a system H>0; H<0 when system loses heat to the surroundings; H=E+PV

Entropy

a measure of disorder or randomness in the system; ordered state is low-entropy; disordered state is high-entropy; systems tend to proceed from ordered to disordered states; change for system+surroundings is unchanged in reversible processes and positive for irreversible processes

Second Law of Thermodynamics

systems tend to process from ordered to disordered states; entropy change for system + surroundings is unchanged in reversible and positive for irreversible; all processes proceed towards equilibrium

Third Law of Thermodynamics

Why is absolute zero important entropy of any crystalline, perfectly ordered substances much approach zero as the temp approaches 0K

Gibbs Free Energy

Thermodynamic function that interrelates enthalpy, temperature, and entropy (G=H-TS); G=0 reaction is at equilibrium; G<0 reaction proceeds as written; G>0 reaction proceeds in opposite direction

What can Thermodynamic Parameters tell us About Biochemical Events

a single thermodynamic parameter is not very useful; comparison of several thermodynamic parameters can provide meaningful insight about a process; positive heat capacity change for a process indicates that molecules have acquired new ways to move and store heat energy; negative heat capacity change means that the process has resulted in less freedom of motion for the molecules involved

Enzymes

catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates; provide cells with the ability to exert kinetic control over thermodynamic potentiality; proteins with catalytic function; lower activation energy

Free Energy of Activation

related to the rate constant; absence of enzyme catalysts cause this to be very large

Catalytic Power

ratio of enzyme-catalyzed rate of a reaction to the uncatalyzed rate

specificity

defines the selectivity of enzymes for their substrates

Oxidoreductases

oxidation-reduction reactions

Transferases

transferring functional groups

Hydrolases

hydrolysis reactions

Lyases

bond cleavage other than hydrolysis or oxidation

Isomerases

isomerization reaction

Ligases

formation of bonds with ATP cleavage

Cofactor

non-protein component of an enzyme

Coenzyme

type of cofactor; usually organic molecule

Holoenzyme

active complex of the protein and prosthetic group

Apoenzyme

protein without prosthetic group; catalytically inactive

Kinetics

seeks to determine the maximum reaction velocity that enzymes can attain and the binding affinities for substrates and inhibitors

Velocity

amount of product formed or the amount of reactant consumed per unit time

Rate laws

describe the progress of the reaction, they are a mathematically expression of the relationship between reaction rate and the concentration of the reactants

Differential Rate Law

expresses the rate of the reaction as a function of the change in concentration of one or more reactants over a particular period of time

Unimolecular Reactions

only one component involved; molecularity refers to the number of molecules that must simultaneously interact

Pre-Steady State

the starting period of the reaction; substrate is in excess

Steady State

the concentration of the enzyme-substrate complex as well as the other reaction intermediates remain approximately constant over time

Steady State Assumption

the change in concentration of ES with time is 0; no net change in steady state

Michaelis Mentens Equation

generated an equation to analyze steady state kinetics based on several assumptions; assume the formation of an enzyme substrate complex; assumes that the ES complex is in rapid equilibrium with free enzyme; assumes that the breakdown of ES to form products is slower than formation and dissociation of ES

Km

kinetic activator constant; ratio of rate constants; small means high affinity; big means low affinity

Vmax

theoretical maximum rate of the reaction but is never achieved in reality; is a constant; all enzyme molecules would have to be substrate bound in order for it to be true

Kcat

turnover number; number of substrate molecules converted to product per enzyme molecule per unit time when E is saturated with substrate

Catalytic efficiency

kcat/km; estimate of how efficient the enzyme isl measures how well the enzyme performs when [S] is low

Lineweaver-Burk Plot

double reciprocal plot because the reciprocal of both sides of the MM equation is taken

Hanes-Woolf Plot

smaller and more consistent errors compared to Lineweaver-Burk

Enzyme activity and pH

enzyme-substrate recognition and catalysis are greatly dependent on pH; enzymes have a variety of ionizable side chains that determine their secondary and tertiary structure and also affect events in the active site; eznymes are usually active only over a limited range; effect may be due to effect on Km or Vmax

Enzymatic Activity and Temperature

enzyme rate typically doubles in rate for every rise as long as enzyme is stable and active; higher causes protein to become unstable and denaturation occurs

Competitive Inhibitor

I and S are structurally similar; high [S] can overcome the effects of I; high [S] only ES species will be present; v decreases as 1/v increases; vmax is unaffected

Noncompetitive Inhibition

inhibitor does not bind to the active site; substrate and the inhibitor do not have structural similarity; interact with both E and ES; rarely interact with S and ES

Pure Noncompetitive Inhibition

presence of the inhibitor has no effect on binding of E to S, S and I bind at different sites, Km is unchanged vmax is decreased, pattern is similar to what would happen with decrease [E]

Mixed Noncompetitive Inhibition

binding of I by E influences the binding of S either the cbinding sites are near one another; conformational changes in E caused by I affect S binding; much more common; km and vmax are altered; km/vmax is not constant

Uncompetitive Inhibition

inhibitor only binds to ES; does not bind to the free enzyme; binding site for inhibitor is only available after the substrate is bound; pattern obtained in a Lineweaver-Burk plot is a set of parallel lines

Irreversible Inhibitors

enzyme and inhibitor are bound, usually by covalent bonds kinetic pattern looks like a decrease in [E] similar to noncompetitive; noncompetitive is instantaneous; irreversible is time dependent

Suicide Inhibitors

activates the enzyme, a reactive group is formed, covalent bonding between E+I makes the enzyme inactive E