Chem 4600 1st EditionExam # 2 Study GuideI. Enzymes- protiens that catalyze reactions. Eacha. are uniqueb. catalyze a specific reactionc. speed up reactions up to 1ˣ 10¹⁶II. Enzyme characteristicsa. Specificity- bind to only 1 (or a few) substratesb. Conformational changec. Selectivity- to a specific substrate-IMF’s attract the enzyme to the substrate to bind (= associate)When a substrate bind enzymea. conformational change occursb. the transition state (Tsⱡ) is stabilizedc. An environment for the reaction is createdIII. Cofactors- any molecule that aids in catalysisa. Coenzyme- organic nonprotien molecule that that aid catalysis and binds (associate) with IMF’sb. Prosthetic group- organic nonprotien molecule that aids catalysis and covalently binds (=tethers)* Enzyme with cofactor= Holoenzyme without cofactor= apoenzymeEnzymes Are Classified on the Basis of the Types of Reactions they catalyzeI. Six types of reactions1. oxidoreductases= dehydrogenases ie ADHone substrate is oxidized and one is reduced. Cofactors are usually NAD+ or FAD+2. transferases= transfer a functional group from one molecule to another ie hexokinase3. Hydrolase= use water to break single covalent bonds. 1molecule→2molecules4. Lyases- break/make covalent bonds without water often forming a new double bond or ring structure**The hardest to label5. isomerase- create isomers of the molecule (rearrangement; 1molecule→1 molecule)6. Ligase- make covalent bonds using energy (ATP or GTP)II. Enzyme Kinetics (the how). 1. It answers:a. Know the enzyme purity (is it contaminated with other protiens?)b. the catalytic efficiency (does it work?)c. enzyme specificityd. enzyme inhibition2. Enzymatic activitya. substrates bind to the active siteb. the active site is where catalysis takes placec. the reaction happens in the catalytic domainIII. How substrates bind to the enzyme (2 theories, 1 followed*)1. Lock and Key- Enzyme binds substrate like a lock fits a key2. Induced fit*- the substrate binds the enzyme, but "fits" to accommodate the Ts. a. A conformational change occurs to allow the stabilization of the TsIV. Enzyme catalyis 1. Enzymes speed up a reaction by lowering the activation energy (Ea)E+S↔ES➡E+PE+S-->ES tells how good the affinity is(Km) a. K1 is the association of S to E b. K-1 is the dissociation of S from EES-->E+P tells how productive it is(K2=Kcat) a. Tells the turnover rate (product/time) b. K2 is the dissociation of P2. Rate of Catalysis a. Velocity- the quantity of S that disappear in a specific amount of time. V= St- /- /- /- /- /- /- /- /- /- /- /- /- / - /b. the quantity of P that appears in a specific amount of timeV P t=/=/=/=/=/=/=/=/=/=/=/=/=/= /V. Kinetic Parameters 1. Vmax= max rate enzyme can theoretically achieve / limit=k₂[Etotal]2.Km= [S] at ½ Vmax; half of enzymes bound; [E]=[ES]- shows affinity; a small number = less [S] to needed to get to ½ Vmax= better affinity3. Kcat= turnover #= products/sec ; ES→ E+P4. Kcat/Km= catalytic efficiency (how fast ES complex form). the bigger the number, the better.5. Specific activity- µM product(U)/mg protien-shows how active your protein is, and confirms if your data is correct, and shows purityVI. enzyme inhibitionVII. Cleveland notationsA. Sequential= all the substrates involved are bound to the enzyme before catalysis of the reaction takes place to release the products.1. ordered sequential= order matters(a)2. random sequential= order doesn’t matter(b)B. Double Displacement (Ping-pong)=an enzyme reacts with one substrate to form a product and a modified enzyme, the latter then reacting with a second substrate to form a second, final product(c)VIII-I. Catalytic residues= amino side chains in the active site that1. are involved in the mechanism (act as an acid or a base)2. Involved in altering the pka of a residueA. Acidic residues (OH→ O⁻) that want a charge will decrease pka to be negative no charge will increase pka to be positiveB. Basic residues (NH₃⁺→ NH₂:) that want charge will increase pka no charge will decrease pka3. weaken covalent bonds (ie. destabilize electron rich molecules3. stabilize the TsVIII-II. Proteases- enzymes that hydrolyze peptide bonds ( break w/ water)A. Acid catalysis- a molecule other than water acts as a proton donor (basic aa-K,R,H)/ electronacceptor (metals)B. Base catalysis- a molecule other than water that acts as a proton acceptor ( acid aa, deprotonated carboxylate, cysteine,tyrosine)C. Acid-base catalysis- hydrolysis of a peptide bond with a strong nucleophile and electrophileD. Types of proteases1. Serine protease- serine does nucleophilic attack, then water 2. Cysteine protease- cysteine does nucleophilic attack, then water 3. Acid(aspartic acid)- acid side chains activate water to make it a better nucleophile4. Metalloprotease- metal activate the electrophile. Sometimes the neutrophileE. Carboxypeptidase A- cuts c-terminal residueAFRIDAY--> AFRIDA+YCPA needs Zn+2 to fold and function properly, therefore is a holoprotein.Mechanism: 1. Substrate bind and active site is created 2. Zn make carbonyl carbon a better electrophile (act as an acid)3. Glutamate take a proton from water (act as a base) 4. Leaving group is (N) because Ts is stabilized by tyrosine *Tyrosine donate proton/accept electrons5. Enzyme resets- tyrosine accept proton from glutamate F. Serine protease chymotrypsin- cuts c-terminal side of a hydrophobic residue (W,F,Y,M,L)AFRIDAY→ AF+RIDAYChymotrypsin consits of a catalytic triad containing Aspartic Acid, Histadien and serineMechanism:1. substrate bind and active site created. The large hydrophobic side chain fits in a “pocket”.catalytic triad forms alkoxide “O” = Ser----O⁻2. Nucleoplilic attack by Serine alkoxide, which makes carbonyl O negative (stabilized by the oxyanion hole)2. The tetrahedral intermediate forms, � electrons travel from O-->C-->leaving group(NH)-->histadine2. � bonds reform, and covalently bind the enzyme= acyl enzyme intermediate2. second nuecleophilic attack to release N-terminal of peptide= the shift Nitrogen of histadine (NH connected to a dbl bond) give electrons to water, so water can attack the peptide2. Second tetrahedral intermediate forms2. enyme is regenerated, releasing the C-terminalHistadine in the chymo reaction1. act as a base- accept H+ from serine2. act as an acid-donate H+=1st nucleophilic attack3. act as a base- accept