Lecture 14: Enzymes & Kinetics IIIMichaelis Menton Kinetics and InhibitionMargaret A. DaughertyFall 2004Announcements!Monday 10/11 lecture: starts at 10:15;Taught by Dr. Stephen EverseNo Office Hour/Review Session Tuesday evening.Review sessions Thurs & Fri from 5:30 - 7:00Homework due Friday 10/15 by 7 pm in my office.Key will be posted on Saturday. Late homeworkwill be penalized!Exam II on Monday 10/18. Problems? See me!Announcements!Kinetics lecture notes: Inhibition lecture: reading: CH 13: pages 421-426 CH 14: pages 460-463Due to time constraints:We will not cover enzyme catalyzed bimolecular rxnsor catalytic RNAs or catalytic Abs (and these willnot be on the exam).We will not cover Hb structure & function. :(Outline• Inihibition Kinetics– Reversible•competitive•non-competitive•Mixed non-competitive•Uncompetitive inhibition– IrreversibleDistinguishable bykinetic inhibitionpatternsEnzyme InhibitionInhibitor (I): compound that decreasesthe rate of a catalyzed reactionTwo modes of action:Reversible: I interacts via non-covalent interactionswith EIrreversible: I interacts via covalent interactionswith E (affinity labels; suicide inhibitors)Reversible Inhibition• Competitive Inhibition: I binds to same site as S• Noncompetitive Inhibition: I binds to different site than S• Mixed non-competitive Inhibition: I binds withdiffering affinities to E and ES complex• Uncompetitive Inhibition: I binds only to EScomplexInhibitors interact with the enzymesthrough noncovalent interactions. Theycan easily associate and dissociate fromthe enzyme.Competitive Inhibition•I mimics S for binding;•I reversibly binds intoactive site;• Binding of I prevents Sfrom binding• However, increasing Scan “compete off” I• Results in an apparentincrease in KM for SsubstrateinhibitorCompetitive InhibitionMichaelis-Menton Equation[E]t = [E] + [ES] + [EI]V = Vmax [S]KM(1+[I]/KI) + [S]V = Vmax [S]KMapp + [S]V vs. [ S ] Graph for Competitive Inhibition0.000.050.100.150.200.250.300.350.400.450.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00velocity[ S ][ I ] = 0[ I ] = 1[ I ] = 2Competitive inhibitors alter Km but do not change Vmax.Identification of CompetitiveInhibition• In the presence ofa competitiveinhibitor–KM increase;–Vmax remains thesame;• Inhibition can beovercome byincreasing [S] -hence competitive!Slope = KM/Vmax; Slope increases with [I]• Ribonucleasecatalyses thehydrolysis of thephosphodiesterbond between 2nucleotides;• UpcA is a strongcompetitiveinhibitor ofribonuclease;Example of Competitive InhibitionPhosphonate analogCompetitive inhibitors are not cures: HIV/AIDS THERAPY *Global summary of the HIV and AIDS epidemic in 2003 (July 2004) *Number of people living with HIV globally in 2003: 37.8 million (34.6-42.3 million) *Number of new infections globally in 2003: 4.8 million (4.2-6.3 million) *Number of deaths due to AIDS globally in 2003: 2.9 million (2.6-3.3 million)Number of people cured of HIV <1Number of infections prevented by vaccination <1 from UNAIDS website, Sept. 2004EpidemiologyNorth Africa & Middle East1%High-income countries2% Latin America & Caribbean7%Eastern Europe & Central Asia6% Asia22%Sub-Saharan Africa62 %Total: 10 millionSource: UNAIDS/UNICEF/WHO, 2004Young people (15-24 years old) living with HIV,Young people (15-24 years old) living with HIV,by region, end 2003by region, end 20032004 Report on the Global AIDS Epidemic (Fig 30)From UNAIDS 2004 global report pptHIV Infection• Kills helper Tcells;• B cells can’tefficientlyproliferate inresponse toantigenstimulation.HIV Protease• Characteristics– Aspartyl protease (pepsin family);– Homodimer, 99AAs/monomer;– All β structure;– Active site• Asp from each monomer• Kd = 440 nM; kcat = 6.8 / sec (pepsin400/sec)• Substrates– Hydrophobic, unbranched AAs at the Nterminal side of the bond to be cleaved;– Small peptides (7 AAs).Wlodawer© 2002HIV protease cleaves a poly-proteinPhe ProNHHNONOHNORR'substrate specificityCompetitive Inhibitors - HIV protease inhibitorsRitonavir and saquinavir are used to treat HIV / AIDS.Inhibits HIV protease (aaspartic protease) whichcleaves larger viral proteinprecursors into HIV proteinsneeded for the virusparticle.Phe ProNHHNONOHNORR'NHHNOOHNHHONHONH2ONSaquinavirOHNOOHNHOHNNONSNSRitonavirWhy are AIDS treatments not cures?Problem 1: inhibitors bind reversibly!Natural substrates can compete for activesite!Problem 2: inhibitor clearance - drugs can bemetabolized in the body.Problem 3: HIV mutation rate gives rise todrug resistance.3.4 x 10-5 HIV-1 mutations/base/replication event;1010 HIV-1 replications/day;Thus, 340,000 mutations/base/dayRitonavir•Ki ~ 15 pM;• High (> 105) specificity for HIVprotease over pepsin;• 70% bio-availability– Good absorption–t1/2 ~ 1.5 hrOHNOOHNHOHNNONSNSWhat drugs have been developed for HIV infection?Twenty drugs have been approved for treating individuals with HIV infection. They are called antiretroviral drugs because theyattack HIV, which is a retrovirus.Once inside the cell, HIV uses specific enzymes to survive. The first approved classes of antiretroviral drugs that were approvedwork by interfering with the virus' ability to use these enzymes. They fall into two categories:*Reverse transcriptase (RT) inhibitors. RT inhibitors interfere with an enzyme called reverse transcriptase or RT that HIV needsto make copies of itself. There are two main types of RT inhibitors, and they each work differently.* Nucleoside/nucleotide drugs provide faulty DNA building blocks, halting the DNA chain that the virus uses to makecopies of itself.* Non-nucleoside RT inhibitors bind RT so the virus cannot carry out its copying function.*Protease inhibitors (PI). Protease Inhibitors interfere with the protease enzyme that HIV uses to produce infectious viralparticles..*Fusion inhibitors interfere with the virus' ability to fuse with and enter the host cell.Drugs Approved for HIV InfectionNucleoside/Nucleotide RT Inhibitors Non-nucleoside RT Inhibitors Protease Inhibitors Fusion Inhibitorsabacavir delavirdine ritonavir pentafusideddC nevirapine saquinavir ddI efavirenz indinavir d4T amprenavir 3TC nelfinavir ZDV lopinavir tenofovir atazanavir entricitabine fosamprenavir calcium www.nih.govNon-competitive Inhibition•I binds to a site other thanthe active site• Does not compete with S forbinding to its