Unit II-Structural Features PDF

Unit-2 : Structural features of different classes of proteins Topics: Role of Transcription factors on gene Nature of interaction between p53 and DNA- effect of mutations in the DNA binding domain of p53- Effects of mutations in the oligomerization and Nuclear localization region Str...

Unit-2 : Structural features of different classes of proteins Topics: Role of Transcription factors on gene Nature of interaction between p53 and DNA- effect of mutations in the DNA binding domain of p53- Effects of mutations in the oligomerization and Nuclear localization region Structural elucidation of leucine zipper- Interaction of leucine zipper and DNA Structural elucidation of GPCR- Types of GPCR- Mechanism of activation of GPCR Structural features of serine proteases  What is transcription? Transcribing genetic information from DNA to RNA Replicatio n DNA DNA Transcriptio n RNA Translatio n Protei n  RNA Polymerase Synthesizes RNA from DNA RNA Polymerase I (Pol I)- Synthesizes rRNAs RNA Polymerase II (Pol II)- Synthesizes mRNAs RNA Polymerase III (Pol III)- Synthesizes tRNAs What is transcription factor? Distal to the RNA PolII initiation site, there are different combinations of specific DNA binding sequences each of which is recognized by a corresponding site-specific DNA binding protein. These proteins are known as transcription factor(s). It constitutes a control module Example: TFIID, TFIIA,TFIIB, TBP etc Architecture of a structural gene and The DNA part of each control module can be divided into three main regions, the core or basal promoter elements, the promoter the promoter-proximal elements and the distal enhancer elements  the core or basal promoter elements  TATA box, a DNA sequence that is rich in A-T base pairs and located 25 base pairs upstream of the transcription start site.  The TATA box is recognized by one of the basal transcription factors, the TATA box-binding protein, TBP, which is part of a multisubunit complex called TFIID.  This complex in combination with RNA polymerase II and other basal transcription factors such as TFIIA and TFIIB form a preinitiation complex for transcription.  promoter proximal elements  usually 100 to 200 base pairs long and relatively close to the site of initiation of transcription.  Within each of these elements there are DNA sequences specifically recognized by several different transcription factors  which either interact directly with the pre initiation complex or indirectly through other proteins. distal enhancer elements  Enhancer elements by contrast are short DNA sequences that occur further upstream or downstream from the initiator site than the promoter proximal elements.  Enhancers contain specific sequences recognized by cognate transcription factors.  The remarkable feature of enhancers is their distance from the promoters they control.  They are often a few thousand base pairs distant from the promoter  This means that in eucaryotes transcription is regulated by a series of DNA sequences, each specifically recognized by a DNA-binding protein, dispersed over very long stretches of DNA Schematic model of transcriptional activation  Transcription factor to bind to a specific DNA sequence Transcription factors Transcriptional Activation  The polypeptide chains of the specific transcription factors usually have two different functions: one is to bind to a specific DNA sequence and another is to activate transcription.  Transcription factor TFIID –the link between specific transcription factors and the preinitiation complex.  TFIID contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs.  The knowledge of Transcriptional activation is limited  However, DNA-binding regions are built up from a very limited number of structural motifs and well studied.  Both x-ray and NMR studies gave structural information on complexes of DNA with members of the four most important families of specific transcription factors: a)helix-turn-helix motifs as in procaryotes, and  B)the leucine zipper, c) helix-loop-helix and d) zinc-containing motifs in eucaryotes  p53 Most innocuous and highly cited biological molecule The “p” in p53 stands for protein and “53” indicates a molecular mass of 53 kDa. The p53 protein plays a fundamental role in human cell growth and mutations in this protein are frequently associated with the formation of tumors. p53 maintains the integrity of the genome during cell division by controlling a critical step in the cell cycle by proteins called cyclins and their associated enzymes, cyclin- dependent kinases These kinases are inhibited by another protein, p21, whose expression is promoted by p53. In the presence of p21 the cell cycle is halted before the cell is committed to divide. Presumably, this gives the cell time either to repair damaged DNA or, if it is beyond repair, to initiate programmed cell death (apoptosis). The wild-type protein binds to specific DNA sequences, whereas tumor-derived p53 mutants are defective in sequence-specific DNA binding and consequently cannot activate the transcription of p53-controlled genes  p53 has an N terminal activation domain followed by a DNA-binding domain, while the C terminal 100 residues form an oligomerization domain involved in the formation of the p53 tetramers.  OLIGOMERIZATION DOMAIN FORMS TETRAMER  The 32-amino acid peptide comprising residues 325–356 of the oligomerization domain of p53 is sufficient for tetramer formation.  The structure of each unit is very simple; a beta-strand-turn alpha-helix motif.  The beta strands form an antiparallel two-stranded beta-sheet and the two alpha helices are arranged in an antiparallel fashion- eight backbone hydrogen bonds allow this formation  Two such dimers form the tetramer through mainly hydrophobic interactions between the alpha helices.  The beta strands are outside the tetramer and are not involved in the dimer–dimer interactions.  The arrangement of the four alpha helices is unusual as four alpha helices packed against each other.  Mutations: 1. A mutation of the beta-strand residue Leu 330 to His.-it with the hydrophilic histidine side chain of the mutant destabilizes the core, prevents formation of dimers, and inhibits p53 function. 2. the mutation of a glycine residue in the turn between the beta strand and the alpha helix. This prevents the subunit from folding into the correct structure for dimerization and hence abolishes p53 function. DNA-binding domain of p53 is an antiparallel beta barrel The crystal structure of the DNA-binding domain (residues 102–292) bound to a 21-base pair DNA fragment containing a specific p53-binding sequence. Beta Barrel Motif DNA binding domain is anti-parallel beta-barrel, with protruding loops from anti-parallel beta-barrel Similar to an immunoglobulin fold (7 of the nine 9 strands) This kind of fold is also present in MHC I binding coreceptor in CD4, NF-KB- controls genes related to stress and infection. While the remaining two strands ( 4 and 5) are short and towards the edges  One end of the barrel is closed together The other end is more open and loops are more extended and protruding outside the barrel, this is the end where DNA binds The conformation of two of these loops is maintained by the Zn atom which is bound to two cysteine side chains from one loop and one Cysteine and one Histidine side chain of another loop  Important interactions with:  Major groove  Arg 280 with G-10  K120 from L1 with G-8  Minor groove  Arg 248 from loop L3 with T-12, T-14 Non-specific interactions between sugar & phosphates in DNA and side-chain & main- chain atoms of the protein Two loops and α helix are involved in the interaction Minor grove interactions at the A-T region are necessary for the minor groove to tightly pack itself with the L3 loop to p53 y compressing the minor groove and causing distortions in DNA Tumorigenic Mutations in the DNA Binding Domai Region  One thousand tumor-causing point mutations of p53 in the light of its structure  R248 mutation–30 percent of p53 mutation related cancer.  Mutations that alter the interaction between L2 and L3  Role of Arg273 with T11’hydrogen bond interactions  Mutations of Arg 280 are found in 2.1% of p53-induced tumors.  L2 (Cys 176 and His 179) and the two side chains from L3 (Cys  238 and Cys 242) mutations can distort L3 loop and affect DNA binding  Leucine Zipper- Structural elucidation of leucine zipper  Leucine zippers are a dimerization domain of the bZIP (Basic-region leucine zipper) class of eukaryotic transcription factors.  The bZIP domain is 60 to 80 amino acids in length with a highly conserved DNA binding basic region and a more diversified leucine zipper dimerization region.  Leucine Zipper Motif ❑ First recognized in yeast transcription factor,GCN4 ❑ Mammalian Transcription factor, C/EBP ❑ proto-oncogenic transcription factors: Fos, Jun and Myc ❑Linear amino acid sequences when plotted in a helical wheel, a remarkable pattern of Leucine residues ❑ Around 30 residues form a modular arrangement of 7 aa residue and the 4th residue (d)always is leucine  First residue usually is hydrophobic (a)  peptide dimerizes and forms two parallel coiled-coil alpha helices with a helical repeat of 3.5 residues per turn, so that the interaction pattern of side chains between the helices repeats integrally every seven residues  a & d position = forms a hydrophobic core region with the leucine residues facing each other  Side chain outside the core (e & g) are frequently charged and can either promote or prevent Dimer formation  The side chains that are immediately outside this core (positions e and g) are frequently charged and can either promote dimer formation by forming complementary charge interactions between the monomers, or  The e and g position amino acids can prevent dimer formation by the repulsion of like charges Dimer: 1. Homodimer- Same transcription factors. 2. Hetero dimer: Two diffferent transcription factor. Example: Fos/Jun heterodimer found in AP1 (Active gene regulating protein 1)- responsible for cell proliferation Jun- Can form both homo and hetero dimer Fos- Can not form homo dimer. As they can not form homodimer, it is not able to bind to DNA all by itself WHY? Answer: Strong charge repulsion of 5 glutamic acid residue in e &g position with no compensating positive charge. ❖Fos can form hetero dimer with Jun due to the complementary positive charges in the e & g position of Jun ❖ Hetero dimer formation facilitates repertoire of DNA binding specificities Two types of monomer– 3 distinct DNA binding specificities Three types of monomer- 6 distinct DNA binding specificities (c)- Fos, (d) - Jun  The heterodimer of Fos- Jun binds to DNA with the same target specificity as the Jun homodimer and with an affinity to the AP1 binding site that is 10 times higher.  Ability of the leucine zipper proteins to form heterodimers greatly expands the repertoire of DNA-binding specificities that these proteins can display Three distinct DNA-binding specificities could, in principle, be generated from two types of monomer  DNA Binding region- leucine zipper ⮚ GCN4-Yeast ⮚ basic region-leucine zipper (b/Zip family) of transcription factor ⮚ Monomer of GCN4 is 281 aa ⮚Binds to promoter regions of more than 30 genes involved in amino acid biosynthesis like during response to amino acid starvation ⮚ Dimerization and DNA binding domains are in two different regions Basic region and C terminal Leucine Zipper region which in total is 55 aa ⮚ DNA recognition region of GCN4 similar to Fos/Jun heterodimer of AP1 ⮚Basic region: about 20 aa long- Eight charged residues, mainly Arg which are involved in DNA Binding ⮚These are disordered in solution in the absence of DNA ⮚GCN4 b/zip region complexed with a DNA fragment of 20 base pairs containing the pseudo- palindromic nucleotide sequence ⮚The dimeric GCN4 molecule is therefore able to bind to the two half-sites in both these DNA recognition sequences even though they have spacer regions of different lengths. (Figure b and c)  Each monomer of the GCN4 fragment forms a smoothly curved, continous alpha helix (see Figure a).  The leucine zipper region of the monomers packs into a coiled coil, where two alpha helices diverge from the dimer axis in a segment comprising the junction between the leucine zipper and the basic regions.  This fork creates a smooth bend in each alpha helix which displaces the basic regions away from the dimer interface so that they can pass through the major groove of DNA, with one alpha-helix on each side of the DNA  Each basic region binds to one half-site with numerous contacts to the DNA, and the structure looks like alpha-helical forceps gripping the major groove of DNA (see Figure b and c)  GCN4 binds to DNA with sequence specific and nonspecific contacts. 4 aa side chain form sequence-specific contact with bases. Asn 235-strictly conserved, is at the centre of interaction area. The side chain of Asn (N) forms 2 H-bonds. Oxygen atoms accepts a H-bond from a N-atom of base C2, & N atom of Asn 235 donates a H-bond to oxygen atom of T3. For this the α helix of GCN4 basic region lies deeply in the major groove. Specifies two of 4 bases in each half site. N235 lies in Hydrophobic pocket along side methyl side chains of Ala 238 &239. A238&239 forms hydrophobic interactions with methyl grps of T3 and T1 Methylene grp of Ser 242-methyl grp of T3. Arg 243 in one monomer donates H-bond to Guanine of G-C bp in bidentate manner. Arg 243 in second monomer forms nonspecific H- bonds to PO4 oxygen atoms in the central region.  The positions of the two alpha helices in the major groove of the DNA and the side chain contacts to bases T1, C2, and T3 are the same in the two half-sites. A number of basic and polar residues donating hydrogen bonds to phosphate oxygens of the DNA backbone. All the eight positively charged residues of the basic region are involved in these contacts, which are conserved between the half-sites The only exception is near the center of the DNA binding region where depending on the spacer size- there could be non-specific interactions if 1 bp is present or specific conserved interactions if 2 bps are present GPCR  Signal Transduction Signal transducing receptors------ Plasma membrane proteins Transmit the signal Elicit a specific response A cascade of enzymatic reaction given rise to many different effects within the cell-like gene expression Binds to extracellular molecules : Growth factors : Hormones : Neurotransmitters Three classes 1. Ion channel linked receptors 2. G protein linked receptors Contains an extracellular domain that 3. Enzyme linked receptors Recognizes specific molecular signals  Signal Transduction receptor Extracellular domain Recognizes specific molecular signal Transmembrane domain Through which signal Is transmitted Intra cellular domain Produces a response Limited number of domains- protein molecules with different functions have been evolved –either by the accumulation of point mutation or by gene shuffling No three dimensional structure is 1. Large size available 2. Membrane-bound 3. Too large to solve by NMR Growth hormone Receptor extracellular domain Amplification of signal by G protein and Protein tyrosine kinase linked Intracellular receptors response G Proteins coupled receptors: an Amplifier Transmembrane domain with six helices Signal transmitted to intracellular domains are amplified by amplifiers called G proteins G protein binds to the Guanine nucleotides and hence named as G proteins Acts as a molecular switch 1. G protein + GTP active state 2. G protein +GDP Inactive ⮚ Slow GTPase activity ⮚ G Proteins + RGS (Regulators of GTP Hydrolysis) Switch off the gene activation ⮚ When in the active GTP-bound state, the G protein can activate many downstream effectors, greatly amplifying the signal, before RGS binds and the signal is switched off.  ❑ Heterotrimers a. Alpha b. Beta c. Gamm a ⮚ When binds to the GTP----- 1. Alpha Dissociates 2. BetaGamma ❑ 1000 different genes code for this receptors ❑ Several Apha, Beta and Gamma subunits---forming different G Proteins - allowing cells to respond to a wide variety of external signals ❑ It is the alpha subunit that contains the GTPase activity.  Inactive state::: Gα-GDP-GβGγ Signals Receptors external signal passes domain through the membrane G Protein activated Cytosolic domain to become activated by conformational change Released and dissociation of Gα-GTP ⮚ All three species that exist in the cell—Gα, GβGγ, GαGβGγ-are consequently attached to the cell membrane through the lipid modification of subunits alpha and gamma ⮚ When GDP is bound to Gα, it forms a heterotrimeric complex with GβGγ but when GTP is bound, Gα remains monomeric. ⮚ As long as the ligand remains on the extracellular domain of the receptor, its cytoplasmic domain can continue to trigger the production of active Gα–GTP molecules. ⮚ Second messenger molecule: a substance whose release within a cell is promoted by a hormone and which brings about a response by the cell. ⮚ GTPase activity determines the length and the time that the signal remains on ⮚ Failure to turn off GTPase activity: Gα –GTP remain active Consequence: Chlolera toxin prevents Gα – GTP breakdown continue excretion of Na and water into the gut. Ras proteins and the catalytic domain of Ga have similar three-dimensional structures  RAS: small GTPases:egulators of signal transduction processes leading to cell multiplication and differentiation  Example: KRAS, NRAS, and HRAS  They are molecular switch activated in response to the protein tyrosine kinase receptor  Ras – Gα similar function- molecular switches  GTPase activity is nil in Ras - GAP(GTPase-activating protein) is required to act on Ras for break down of GTP to GDP  25% tumor cells produce mutant Ras protein not regulated by GAP as these mutant Ras molecules remain bound to GTP and activated, and this leads to uncontrolled cell growth  Structural details of Ras  α/β type  six beta strands – five parallel and five alpha helices positioned on both sides of the beta sheet  Loop regions that connect the beta strands with the alpha helices form the binding pocket.  Mg2+ is required both for proper positioning of the gamma phosphate  Also for weakening the P–O bond that is split during catalysis, as well as for maintaining the stability of the guanine nucleotide complex  5 out of 6 loops in Ras involved in GTP binding site ⮚ 3 of these loops, G1 (10 – 17), G3 (57 – 60) and G4 ( 116 – 119) conserved in all GTP binding proteins ⮚ G1 – for proper positioning of the phosphate groups – binds to the α and beta phosphate nucleotide- P loop  G3 – link subsites of Mg2+ binding and the γ phosphate  G4 – recognition and binding of guanine nucleotide  Loops G4 and G5 are involved in recognizing and binding the guanine base of the nucleotide. ❑Two switches – conformational changes on activation Switch I – G2 Thr binds Mg2+ and involved in structural switching and GTP hydrolysis Switch II – G3 and alpha 2 G1 - G-X-X-X-X-G-K-S/T G3 - D-X-X-E G4 -N-K-X_D Transducin to study the GTP hydrolysis by G-alpha  G – Protein associated with rhodopsin Effector enzyme – cAMP phosphodiesterase  Much larger than Ras and has two domains – GTPase domain similar to Ras and alpha-helical domain with a unique topology  The linker region ensures that the exchange of guanine nucleotide is regulated.  The linker region has one large alpha helix (28 residues) with 5 supporting small helices acting as a gate.  Ga has three switch regions  Details of the conformational differences between these two states of G-alpha by comparing high-resolution structures of G-alpha complexed with GDP (resting form) and GTP-gammaS (active form).  There are three switches -Two of which are the same as switch regions I and II described for the Ras structure, while the third, switch region III, is another loop region linking ß4 with α3  Four water molecules as well as the side chain oxygen atom of a serine residue from the P-loop and one oxygen atom from the ß- phosphate bind to Mg2+ in the GDP structure  Two of the water molecules are replaced in the GTP structure by a threonine residue from switch I and an oxygen atom from the gamma phosphate Structural features of serine proteases  Serine Proteases Enzymes Increases the rate of the reaction by decreasing the activation energy by stabilizing transition states Turnover number and specificity constant Kcat= Turnover number and kcat/ KM= Specificity constant In simple reactions- kcat is the rate constant for the chemical conversion of the ES complex to free enzyme and products the specificity constant describes the specificity of an enzyme for competing substrates.  Lowering the activation energy Enzyme bind to the substrate more tightly in transition state than its ground state  Lowering the activation energy Enzyme bind to the substrate more tightly in transition state than its ground state  The activation energy for the conversion of ES complex to E and P is lower if the Enzyme binds more tightly to the substrate in its transition state compared to the Normal state. The higher affinity of the enzyme for the transition state makes the transition energetically favorable and thus decreases the activation energy. If the enzyme were to bind the unaltered substrate more strongly than the transition state, the decrease in binding energy on the formation of the transition state would Increase the activation energy and catalysis would not be achieved. Question: Enzyme active site should be complementary to the transition state of the substrate than to the normal structure------explain. catalytically advantageous for the enzyme’s active site to be complementary to the transition state of the substrate rather than to the normal structure of the substrate. This decreases the activation energy  Four classes of Proteases:  Aspartyl  Cysteinyl Based on active site residue  Serine  Metallo Serine proteinases: Most extensively studied by X-Ray Crystallography and Kinetic methods Cleave peptide bonds within a polypeptide to produce two smaller peptides Two step reaction : 1. Covalent bonds between C1 of the substrate and –OH of the serine of the enzyme. 2. Deacylation: The acyl-enzyme intermediate is hydrolyzed by a water molecule to release the second peptide products with a complete carboxy terminal and restore the serine -OH of the enzyme  The first step produces a covalent bond between C1 of the substrate and the hydroxyl group of a reactive Ser residue of the enzyme.  Production of this acyl-enzyme intermediate proceeds through a negatively charged transition state intermediate where the bonds of C1 have tetrahedral geometry in contrast to the planar triangular geometry in the peptide group.  During this step the peptide bond is cleaved, one peptide product is attached to the enzyme in the acyl-enzyme intermediate, and the other peptide product rapidly diffuses away.  In the second step of the reaction, deacylation, the acyl-enzyme intermediate is hydrolyzed by a water molecule to release the second peptide product with a complete carboxy terminus and to restore the Ser-hydroxyl of the enzyme  This step also proceeds through a negatively charged tetrahedral transition state intermediate Structural features : Catalytic traid consisting of Asp-His-Ser. Provide a general base Histidine that accept proton from the –OH group Of Serine. Oxyanion hole: Making hydrogen bond with the charged oxygen atom attached to the C1 of the substrate Non specific binding site: Polypeptides bind to the enzyme non specifically, which forms hydrogen bonds in a short antiparallel beta-sheet with main-chain atoms of a loop region in the enzyme Specificity pocket: Preferred a particular side chain before Scissile bond Chymotrypsin super family: 1. Chymotrypsin 2. Trypsin 3. Thrombin 4. Elastin Bacterial serine proteases Mammalian serine proteinases and bacterial serine Proteinases (Subtilisin) have different sequence, structure but the have the same active site architecture ---indicates convergent evolution 1.Catalytic triad 2.Oxyanion hole 3.Substrate binding Almost in the same position in all cases Structural solution to achieve a particular catalytic mechanism  Chymotrypsin structure: Chymotrypsinogen to Chymotrypsin 254 aa 14-15 and 147-148 excised 3 polypeptides held together by disulphide bridge Two domain with similar structure 120 aa acids each Antiparallel beta barrel type with six beta stand Greek key motif (1-4) followed by a hairpin motif  Active site: Crevice of domain1 and domain 2 Domain 1- Histidine 57 and Asp 102 Domain 2 – Ser 195  Role of substrate specificity pocket Different proteinases cleaves the same substrate (polypeptide chain specifically at different residues Chymotrypsin – Next to aromatic residues Trypsin – next to positively charged residues Elastase – small hydrophobic residues Structural details imparts this specificity Chymotrypsin- Wide pocket, Trypsin- because of Asp, positively charged Lys and Arg large aromatic side chain accommodated,  Mutational effect on active site of Trypsin Gly 216-Ala 216= Ala 216 mutant would displace a water molecule at the bottom of the specificity pocket that in the wild type enzyme binds to the NH3+ group of the substrate Lys side chain. The extra CH3 group of this mutant is not expected to disturb the binding of the Arg side chain. Km for Lys substrates would increase and therefore kcat/Km would decrease more for Lys than for Arg-containing substrates. values for both Lys- and Arg-containing substrates 6 - Leads to decrease in Km.  Gly 226-Ala 226 = The Ala 226 substitution would introduce a methyl group in the region where the end of the substrate’s side chain binds and would therefore be expected to accommodate Lys better than Arg, since the latter has a longer and more bulky side chain. the Km for an Arg-containing substrate would be larger (less favorable binding) and the Km for Lys would be essentially unaltered. The specificity constant, kcat/Km, would decrease more for an Arg-containing substrate than for one with Lys Gly216 Gly 226-Ala216Ala22= For the double mutant where both Gly 216 and Gly 226 are changed to Ala, one would predict an increase in the Km values for both Lys- and Arg-containing substrates. In all of the mutations - the largest changes occurred in the catalytic rates while the mutations were done to change the specificity  Effect of mutation at Asp 189 Asp 189 at the bottom of the surface, interacts with Lys and Arg of the substrate this is the basis of the cleavage A mutation ASP189-Lys189 would have abolish binding with Lys and Arg rather it will bind to the Asp. The result was surprising as Asp-Lys mutant was totally inactive with both Asp and Glu substrate. As expected , inactive for both Lys and Arg. Surprisingly active against Phe and Tyr with the same turnover number as wild-type tyrosine. It showed 5000 fold increase in Kcat/Km with Leu substrate The three-dimensional structure of this interesting mutant has not yet been determined  Bacterial substilisin Isolated from bacilli Added in the detergent to remove proteinacious dirt First engineered protein patented in USA 1988 Single polypeptide chain of 275 aa No sequence similarity with Chymotrypsin but having same active site architecture Alpha beta structure - 5 parallel beta strand and four helix -two on each side of the parallel beta sheet Catalytic triad- Asp32, His 64, Ser 221 Oxyanion hole- Asn 155 Specificity pocket- Hydrophobic aa nonspecificity- antiparallel sheets Inhibitor- Eglin  Structural anomaly of Bacterial subtilisin 3 β α β motif, all are right handed β2 αB β3- Left handed β23 αC β4 - Right handed β4 αD β5 - Right handed His 64, which is part of the catalytic triad, is in the first turn of helix αB This helix would be on the other side of the beta-sheet, far removed from the active site if the motif β2 αB β3 were right-handed. So to produce a proper catalytic triad of Asp 32, His 64, and Ser 221, helix αB must be on the same side of the beta-sheet as Ser 221-the motif has evolved to be left-handed.  Mutational effect of Subtilisin Ser 221 to Ala 221= Kcat and Kcat/Km reduced---106 ????? Answer: Mutaion at Ser 221 stops the covalent binding with the substrate and hence abolish the reaction mechanism His 64-Ala 64 and Asp32 to Ala 32 ------ Both have no effect on the catalytic Reaction rate since the catalytic triad is no longer in operation. The enzyme showed activity 1000 times faster than non-enzymatic rate Active site (not the residue) binds to the substrate tightly in its transition state than its initial state  Substrate assisted catalysis His 64- Ala 64 Same effect that is decrease the Ser 221- Ala 221 reaction rate by 106 Asp 32 to Ala32 An essential group that is lacked by a mutant enzyme can be replaced by Similar group from the substrate  One consequence of substrate-assisted catalysis is that the mutant enzyme is highly specific for substrates containing the essential group.  The His 64–Ala mutant of subtilisin, for example, has a specificity factor (ratios of kcat/Km) of about 200 for substrates containing histidine.  experiments confirmed this prediction and showed that the mutant His 64–Ala catalyzes hydrolysis of a peptide substrate about 400 times faster when the peptide has histidine at the appropriate position in its sequence.  The single mutation Asp 32–Ala reduces the catalytic reaction rate by a factor of about 104 compared with wild type. This rate reduction reflects the role of Asp 32 in stabilizing the positive charge that His 64 acquires THANKYOU