Biochem Test 2

Chapters: 6, 7, 8, 10, 11, 12, 13, 33, and some 34-35

Serine Proteases

synthesized as zymogens in the pancreas then activated by selective proteolysis

selective proteolysis

activates serine proteases

Digestive serine proteases

Trypsin, Chymotrypsin, Elastase

zymogens

inactive enzyme precursers that must be covalently modified to become active

trypsinogen, chymotrypsinogen, proelastase

zymogens of trypsin, chymotrypsin, elastase

enzyme inhibitors

regulates pancreatic zymogens

enzyme cascades

rapid signal amplification

enteropeptidase

activates trypsin

elastase

cleaves on the carbonyl side of AA with small uncharged side chains

structure

dictates function

chymotrypsin

active, has a binding pocket

chymotrypsinogen

inactive, AA residues bonded to block the binding pocket, cleavage between residues 13/14, 15/16, and 146/147, opens binding pocket by turning Asp194 outward

Binding site of chymotrypsin

binding site of trypsin

binding site of elastase

binding site

causes substrate specificities due to small structural differences in active site binding cavities

catalytic triad

Asp, His, Ser

a-chymotrypsin mechanism: scissile peptide bond

carbonyl carbon positioned next to the oxygen of Ser-195

a-chymotrypsin mechanism: specificity pocket

binds R1 group holds substrate in place

a-chymotrypsin mechanism: enzyme substrate complex

1. substrate binding compresses Asp and His

2. imidazole removes H+ from Ser

3. nucleophilic O of Ser attacked carbonyl of substrate

4. first tetrahedral intermediate formed

a-chymotrypsin mechanism: tetrahedral intermediate 1

1. substrate C-O double bond changes to single bond

2. negatively charged O moves to oxyanion hole and H-bonds to NH of Gly and Ser

3. imidazolium of His donates H+ to N of scissile bond yielding cleavage

a-chymotrypsin mechanism: acid-base and covalent catalysis

1. carbonyl from remaining peptide forms covalent bond with enzyme yielding acyl-enzyme intermediate

2. peptide product with new amino terminus leave active site

a-chymotrypsin mechanism: hydrolysis

1. final substrate, H2O enters binding pocket

2. His abstracts H+ from H2O

3. Nucleophilic O of OH- reacts with carbonyl of enzyme-acyl intermediate

a-chymotrypsin mechanism: tetrahedral intermediate 2

1. His imidazolium ion donates H+

2. formation of second tetrahedral intermediate

3. stabilized by oxyanion hole

a-chymotrypsin mechanism: product 2

second polypeptide product formed which has new carboxy terminus

a-chymotrypsin mechanism: enzyme and product

1. final polypeptide product leaves active site

2. enzyme can now cleave a new polypeptide

Nucleophilic substitution reactions

ionic reaction where both electrons stay with one atom→ ionic intermediate + leaving group

contain a nucleophile and an electrophile

Direct displacement

two molecules react to form a 5 group transition state

formation of tetrahedra intermediate

type of nucleophilic substitution

cleavage reactions (both electrons)

most common, both electrons stay with one atom

ex: formation of carbanion or carbocation

cleavage reactions (one electron)

less common, one electron remains with each product

formation of free radicals

Carbanion

made from cleavage reactions

carbon retains both electrons

carbocation

made from cleavage reactions

carbon looses both electrons

oxidation reduction reactions

addition of O, removal of H, removal of electrons

electrons transferred between two species

oxidizing agent

gains electron (is reduced)

reducing agent

donates electron (is oxidized)

enzymes

lower activation energy of reactions

substrate binding

enzymes position substrates for reaction, formation of transition state more frequent and lowers activation energy

transition state binding

transition state bound more tightly than substrates, lowers activation energy

Binding modes of enzymes

proximity effect and transition state stabilization, increase reaction rate 10,000 to 100,000 fold

proximity effect

collects and positions substrates into active site

1. reduces their degrees of freedom

2. results in a large loss of entropy

3. enhanced concentration of substrates predicts rate acceleration

transition stat stabilization

transition state binds more tightly than substrates

reactions of carboxylates with phenyl esters

increased rates are seen when the reactants are held more rigidly in proximity

thermodynamic pit

formed due to excessive enzyme substrate complex stabilization, little or no catalysis

Km

substrate dissociation constant, indicate weak binding to enzymes

Serine protases active site

Asp-102, His-57, Ser-195 are arrayed in a hydrogen bonded network

cofactors

required by some enzymes for activity

essential ions

type of cofactor, mostly metal ions

coenzymes

type of cofactor, organic compounds, act as group (ex H/ electrons) transfer reagents

Apoenzyme + cofactor → holoenzyme

inactive protein only → activated

metal ions

participate in catalysis, tightly bound, participate in binding of substrate at the active site

metabolic coenzymes

can be synthesized within the body

vitamin derived coenzymes

cannot be synthesized within the body

activator ions

type of essential ion, loosely bound

includes: Ca++, K+, Mg++, Mn++

metal ions of metalloenzymes

type of essential ion, tightly bound

includes: Fe-5 center, zinc, copper, cobalt

cosubstrates

type of coenzyme, loosely bound

includes: ATP, SAM, UDP-sugar, NAD+/NADP+, tetrahydrofolate, CoA, ubiquinone, protien coenzymes

altered during reaction and regenerated by another enzyme

prosthetic groups

type of coenzyme, tightly bound

includes: FMN/FAD, TPP, PLP, biotin, adenosyl / methyl cobalamin, lipoic acid / lipoamide

remain bound to the enzyme during the reaction and may be covalently / tightly bound to enzyme

pyruvate dehydrogenase complex

requires 5 coenzymes, in eukaryotes has 102 subunits

coenzyme: thiamine pyrophosphate (TPP)

Enzyme: pyruvate dehydrogenase

coenzyme: flavin adenine nucleotide (FAD)

Enzyme: mono amine oxidase

coenzyme: nicotinamide adenine dinucleotide (NAD+)

Enzyme: lactate dehydrogenase

coenzyme: pyridoxal phosphate (PLP)

Enzyme: glycogen phosphorylase

coenzyme: coenzyme A (CoA)

Enzyme: acetyl CoA carboxylase

coenzyme: biotin

Enzyme: pyruvate carboxylase

coenzyme: 6’-deoxyadenosyl cobalamin

Enzyme: methyl malonyl mutase

coenzyme: tetrahydrofolate

Enzyme: thymidylate synthase

metal: Zn2+

Enzyme: carbonic anhydrase

metal: Mg2+

Enzyme: EcoRV

metal: Ni2+

Enzyme: urease

metal: Mo

Enzyme: nitrogenase

metal: Se

Enzyme: glutathione peroxidase

metal: Mn2+/3+

Enzyme: superoxide dismutase

metal: K+

Enzyme: acetoacyl CoA thiolase

SAM (s-adenosylmethionine)

donor of methyl groups for most biosynthetic reactions (ex synthesis of hormone epinephrine from norepinephrine)

Vitamins

organic substance required in trace amounts for a number of essential biochemical reactions

cannot be made by an organism instead obtained from nutrients

vitamin: ascorbate (C)

disease: scurvy

vitamin: nicotinic acid (B3)

disease: pellagra

vitamin: riboflavin (B2)

disease: growth retardation

vitamin: pantothenate (B5)

disease: dermatitis in chickens

vitamin: thiamine (B1)

disease: beriberi

vitamin: pyridoxal (B6)

disease: dermatitis in rats

vitamin: biotin

disease: dermatitis in humans

vitamin: folate

disease: anemia, spina bifida

vitamin: cobalamin (B12)

disease: pernicious anemia

vitamin c

not coenzyme, reducing reagent for hydroxylation of collagen

thiamine (B1)

thiamine pyrophosphate (TPP)

typical reaction type: aldehyde transfer

riboflavin (B2)

flavin adenine dinucleotide (FAD)

typical reaction type: oxidation-reduction

pyridoxine (B6)

pyridoxal phosphate

typical reaction type: group transfer to or from amino acids

nicotinic acid (niacin B3)

nicotinamide adenine dinucleotide (NAD+)

typical reaction type: oxidation-reduction

pantothenic acid (B5)

coenzyme A

typical reaction type: acyl-group transfer

biotin (B7)

biotin-lysine adducts (biocytin)

typical reaction type: ATP-dependent carboxylation and carboxyl-group transfer

Folic acid (B9)

tetrahydrofolate

typical reaction type: transfer of one-carbon components; thymine synthesis

cobalamin (B12)

5’-deoxyadenosyl cobalamin

typical reaction type: transfer of methyl groups; intramolecular rearrangements

NAD+

NADH

FAD

FADH2

CoA

thiamine pyrophosphate (TPP)

pyridoxal phosphate