Biochem Exam 2

Define and distinguish general acid-base catalysis versus covalent catalysis

Acid-base catalysis: acceleration of a reaction achieved by the transfer and acceptance of H+ OR the generation of OH- in solution from water

Covalent catalysis: part/all of a substrate binds covalently to the enzyme to form an intermediate (EX) before transferring to a second substrate

AX + E ⇌ EX + A EX + B ⇌ BX + E

Discuss metal ion catalysis

Essential metal ions that act as inorganic cofactors in enzymes. They bind tightly as electrophilic catalysts, can generate nucleophiles, and participate in substrate binding

Define what a pH curve is and how it informs one about amino acids that function in the catalytic site of an enzyme

A pH curve is the enzyme reaction rate compared to pH levels. Inflection points can indicate pKa values of the ionizable catalytic residues.

Distinguish reversible versus non-reversible inhibition

Reversible inhibition: the inhibitor binds non-covalently, is temporary, and easily dissociates from the enzyme. Includes competitive, uncompetitive, and noncompetitive inhibition

Irreversible inhibition: the inhibitor binds, stays attached, and modifies amino acids. Permanent and includes active-site directed and suicide inhibitors

Define and distinguish competitive, noncompetitive and uncompetitive reversible inhibition

Competitive: inhibitor binds to the free enzyme (E) at the substrate binding site, competing with the substrate

Uncompetitive: inhibitor only binds to the enzyme-substrate complex (ES), decreasing both Vmax and Km

Noncompetitive: inhibitor binds to both E and ES at a site distinct from the active site; decreases Vmax while Km remains unchanged

Describe a Lineweaver-Burk plot and explain how to use it to derive information about enzyme inhibitors

A double-reciprocal plot with 1/v0 vs. 1/[S]. Y-intercept = 1/Vmax and X-intercept = -1/Km. Slope = Km/Vmax

Competitive intersects on the y-axis

Uncompetitive gives parallel lines

Noncompetitive intersects on the x-axis

Describe how irreversible inhibitors can be used to map active sites of enzymes including the use of affinity labels

Irreversible inhibitors covalently modify specific active-site residues

Affinity labels resemble the substrate to target the active site and covalently react with catalytic amino acids, allowing researchers to identify/map catalytic residues

Describe site-directed mutagenesis and how it can be used to study enzymes

Site-directed mutagenesis is a precise alteration of a specific amino acid codon in an enzyme’s gene to test its role in binding or catalysis

Define what the catalytic site and catalytic residues are

Catalytic sites: a specialized region containing polar/ionizable amino acids + H2O molecules that undergo chemical changes during catalysis

Catalytic residues: the specific amino acid side chains that undergo/interact with chemical changes

Distinguish the substrate binding site(s) and catalytic site of enzymes

Substrate binding sites are pockets that recognize and bind a substrate via non-covalent interactions

Describe what is meant by enzyme mechanism and give some examples

Triose Phosphate Isomerase (TPI): catalyzes aldehyde-ketone interconversion between DHAP + G3P via a proton-shuttling mechanism involving an enediolate intermediate utilizing Glu-165 and His-95

alpha-Chymotrypsin: serine protease uses a catalytic triad (Asp-102, His-57, Ser-195) to perform acyl-enzyme covalent catalysis and general acid-base catalysis with tetrahedral intermediates

Explain what serine proteases are and define their specificity

Serine proteases are digestive enzymes synthesized in the pancreas as an inactive zymogen and activates by proteolysis in the small intestine

• Chymotrypsin: deep hydrophobic pocket accepting a large aromatic chain (Tyr, Phe, Trp)

• Trypsin: deep pocket with a negatively charged Asp at the bottom accepting positively charged Lys/Arg

• Elastase: shallow pocket restricted by bulky Val and Thr side chains, accepting small uncharged amino acids (Ala, Gly)

Describe the three levels of enzyme regulation

1. Genetic Control: regulation of enzyme concentration via transcription/translation synthesis and degradation

2. Covalent modification: activation/inactivation by covalent changes or zymogen cleavage

3. Allosteric regulation: reversible non-covalent binding of effector molecules at allosteric sites

Distinguish the binding mode and chemical modes of enzyme catalysis

Binding mode: proximity effect + transition-state stabilization, providing 10^4 - 10^5 fold rate

Chemical modes: acid-base and covalent catalysis, providing 10- 100-fold rate enhancement

Describe what information an energy diagram gives you about enzyme catalyzed reactions

An energy diagram can illustrate ground states, intermediate energy walls, and activation energy barriers

Compare and contrast proximity effect and transition state stabilization in regards to enzyme catalysis

Proximity effect increases effective reactant concentration to promote transition-state formation by 10^4-10^5 fold-rate

Transition-state stabilization: the enzyme binds transition states 10^10-10^15 times more tightly than substrates, directly lowering activation energy and overall leading to a 10^4-10^5-fold rate increase

Describe the possible functions of the seven amino acids with ionizable side groups in catalytic sites

Aspartate - COO^- allows cation binding/proton transfer

Glutamate - COO^- allows cation binding/proton transfer

Histidine - imidazole provides general acid-base proton transfer

Cysteine - SH provides covalent binding of acyl groups/nucleophile

Tyrosine - phenol allows hydrogen bonding to ligands

Lysine - NH3+ allows anion binding and proton transfer

Arginine - anion binding

Explain the structure, group transferred and type of reaction associated with for each of the major 12 coenzymes

Uh yeah best of luck

Know whether the 12 major essential coenzymes are vitamin-derived or metabolite-derived and whether they are cosubstrates or prosthetic groups

mhm…continue best of luck

For the vitamin-derived coenzymes, know from which vitamin they are derived and where that vitamin-derived structure is in the coenzyme

yep yep

Understand the role of carbohydrates as major recognition and cell-cell interaction determinants

Lymphocyte → lymph nodes

ABO blood → cell-surface antigen

Serves for recognition determinants in cell-cell interaction events

Explain the definition of carbohydrates in regards to polyhydroxyl aldehydes and polyhydroxyl ketones

Polyhydroxyl aldehydes: the carbonyl group is located at the end of the molecule

Polyhydroxyl ketones: the carbonyl group is located internally in the molecule

State the different functions of carbohydrates

• Energy storage

• Metabolic intermediates

• Structural framework

• Extracellular matrix

• Cell-cell recognition + signaling

Define and distinguish the terms: monosaccharides, disaccharides, oligosaccharides, glycans, aldoses, ketoses, glycobiology, glycoconjugate

Monosaccharides: individual monomeric sugar units

Disaccharides: 2 monosaccharides covalently bonded

Oligosaccharides: short chains containing 2-20 linked monosaccharides

Glycan: a term for a carbohydrate polymer

Glycoconjugate: a complex molecule where one or more carbs are linked to a non-carb species such as a protein/lipid

Glycobiology: the study of glycan and glycoconjugates in nature

Discuss D vs L sugars

For sugars containing 1+ chiral centers, this refers to the configuration of the furthest chiral carbon from the aldehyde/ketone group.

D= right

L= left

Understand terms such as triose, tetrose, pentose, hexose, etc.

Triose- 3 carbons

Tetrose- 4 carbons

Pentose- 5 carbons

you get the idea

Define the term epimer and give examples

An epimer is a stereoisomer that differs in configuration at only one specific chiral center

Be able to draw the structure of representative tri, tetra, penta, and hexa- aldoses and ketoses and know their common and scientific names

uh… yeah you got it man

Understand hemiacetals and hemiketals and linear vs cyclic sugars

Hemiacetals: a reaction with an aldehyde group

Hemiketal: a reaction with a ketone group

Linear: chains

Cyclic: well…cyclic

Define the terms pyranose and furanose

Pyranose: a 6-membered ring system

Furanose: a 5-membered ring system

Distinguish an alpha versus beta anomeric linkage in carbohydrates

An anomeric carbon is a carbon atom in a cyclic sugar originally part of the carbonyl group in the open-chain form

Alpha anomeric linkage: the OH group on the anomeric carbon is trans relative to the C6 substituent

Beta-anomeric linkage: the OH group on the anomeric carbon is cis relative to the C6 substituent

Give examples of the conformation of cyclic pentose and hexose

Cyclic pentose: Exist in 10 envelope conformations

Cyclic hexose: adopt 2 chair conformation and 6 boat conformations with bulky hydroxyl groups in equatorial positions (most stable)

Explain and give examples of common biological phosphate, acidic, deoxy and amino sugars

Yep you got it

Know the three letter abbreviation of common biological sugars

Yerp

Explain what a glycosidic bond is and distinguish the terms glucoside vs glycoside

Glycoside: any compound where a carbohydrate provides the anomeric carbon

Glycoside: a specific glycoside where glucose provides the anomeric carbon

A glycosidic bond is a condensation bond formed under acidic conditions between the anomeric carbon of a sugar and an alcohol, amine, or thiol

Describe the structure, common and scientific name and source of the major disaccharides maltose, cellobiose, lactose, sucrose

Yerp

Distinguish reducing end vs non-reducing end of an oligo/polysaccharide

Reducing end: end containing a free, unbounded anomeric carbon

Non-reducing end: the end where the anomeric carbon is locked into a glycosidic linkage

Define the terms aglycone and glycoside

Aglycone: the non-sugar organic group attached to the sugar’s anomeric carbon in a glycoside

Glycoside: any compound containing a glycosidic bond where a carbohydrate provides the anomeric carbon

Distinguish between homopolymer vs heteropolymer

Homopolymer: composed of a single repeating monosaccharide species

Heteropolymer: composed of two or more distinct monosaccharide species

Describe the structure and function and distinguish of the glucohomopolymers starch and cellulose

Starch: plant storage polymer consisting of linear amylose and branched amylopectin.

Cellulose: structural component of plant cell walls consisting of unbranched glucose chains that interact via intra/inter-chain hydrogen bonds to form microfibrils and tough fibers

Define Degree of Polymerization (DP)

The total number of monomeric units in a macromolecule or polymer

Distinguish endoglycanase versus glycosidase

Endoglycanase: cleaves internal glycosidic bonds at random positions within a polymer chain

Exoglycosidase: cleaves monosaccharides or disaccharides sequentially from the non-reducing end of the chain

Explain the difference between alpha-amylase and beta-amylase

Alpha-amylase: an endoglycanase present in salive and pancreas that cleaves random internal alpha-1,4-glucosidic bonds

Beta-amylase: an exoglycosidase that acts from non-reducing ends to release maltose dimers

Describe the structure and function of chitin

Structural homopolymer composed of repeating beta (1→4)-linked N-acetylglucosamine residues. Found in arthropod exoskeletons and fungal cell walls

Explain what glycosaminoglycans and proteoglycans are

Glycosaminoglycans: unbranched heteroglycans composed of repeating disaccharides containing an amino sugar

Distinguish N-linked vs O-linked oligosaccharides and glycoprotein

N-linked: sugar attached via an N-glycosidic linkage to the amide nitrogen of an Asparagine residue within the consensus sequence Asn-X-Ser/Thr (X=any amino acid except Pro)

O-linked: sugar attached via an O-glycosidic linkage to the hydroxyl oxygen linkage to the hydroxyl oxygen of Serine or Threonine residues