Advanced Biochemistry Biophysical Methods
Advanced Biochemistry Biophysical Methods CHEM 200
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Date Created: 09/07/15
Cell Vol 110 665 668 September 20 2002 Copyright 2002 by Cell Press The Path to Perdition ls Paved with Protons Rachel Greenm and Jon R Lorsch Howard Hughes Medical Institute Department of Molecular Biology and Genetics 2Department of Biophysics and Biophysical Chemist School of Medicine Johns Hopkins University 725 N Wolfe Street Baltimore Maryland 21205 Recent studies are beginning to shed light on the mechanism of ribosome catalyzed peptide bond for mation It s Not Just Chemistry It s Biology In 1976 Jeremy Knowles wrote a review article summa rizing some of the reasons why a large proportion of pHdependence studies yields little information about the real pKavalues of ionizing groups in enzymes and still less about the identity of these groups Knowles 1976 The purpose of this review is to summarize recent work on the pH dependence of the peptidyl transferase activity of the ribosome and the molecular basis for this fundamental enzymatic reaction In deference to Knowles however we will proceed with caution The ribosome translates the information contained in a template mRNA into the encoded polypeptide The quot 39 39 quot in quot I Ifquot lUlllldllUll reac tion Figure 1 are two different tRNAs one with the growing peptide chain attached via an ester linkage to its 8 hydroxyl the P site or peptidyl tRNA and the other with a single amino acid esterified to its 8 hydroxyl the A site or acceptor tRNA Peptide bond formation is a seemingly simple reac tion attack of an amine on an ester to produce an amide and an alcohol Figure 1 ln aqueous solution at pH 7 most amines exist predominantly in the ammonium ion RNHg form This species has no free lone pair elec trons on L quot a thu mu t r Figu e 1 step 1 to generate the free amine RNHZ before any reaction with the ester can occur The free amine can then nucleophilically attack the carbonyl carbon of the ester step 2 producing a zwitterionic tetrahedral inter mediate referred to as T Satterthwait and Jencks 1974 In most cases the T intermediate loses a proton to generate the intermediate T step 3 which breaks down to yield the products of the reaction step 4 De pending on the natures of the ester and the amine and on the pH of the reaction either the breakdown of the T intermediate or the attack of the amine on the carbonyl carbon of the ester can be the ratelimiting step in solu tion Satterthwait and Jencks 1974 FU u um I must move a proton must be lost from the ammonium ion to generate the reactive amine in step 1 a proton 3Correspondence ragreenjhmiedu RG jlorschjhmiedu JRL Minireview must be lost from the T intermediate in step 8 and a proton must be picked up by the oxygen atom of the leaving group the ribose of the P site tRNA in step 4 Thus in catalyzing the formation of a peptide bond the ribosome must minimally accommodate the movement of these protons eg through solvent water or actually facilitate their movement general acidbase catalysis f 39 r uestion that mu 1 39 A to understand how the ribosome synthesizes proteins is how it deals with these protons In addition to shuffling protons there are a number of other ways by which the ribosome could promote peptide bond formation The most general mode of ca talysis Sr L k for reaction to occur Jencks 1969 is a strategy that is certainly used by the ribosome given that it has bind ing sites for both the tRNA substrates The ribosome could also stabilize the negative charge that develops on the carbonyl oxygen in the transition state Figure 1 analogous to the role of the oxyanion hole in the serine proteases Furthermore if a negative charge de velops on the hydroxyl oxygen of the ribose leaving L 1 In L Y statically stabilize this charge The challenge is to figure out which of these various strategies are actually em ployed by the ribosome and what the relative contribu tions of each strategy are to the overall rate en hancement here Are Protons Moving in There Somewhere In the late 19603 it was shown that the rate of the peptidyl transferase reaction increases as the pH is in creased from 6 to 8 above which it reaches a plateau Maden and Monro 1968 Rate versus pH curves yielded an apparent pKa for the system of N75 As this value is near the expected pKa for deprotonation of the ammonium form of the nucleophile in the reaction it was possible that it was the titration of this proton that was being observed To check this Rich and coworkers Fahnestock et al 1970 compared the pHrate profiles for ribosomecatalyzed peptide bond formation for an A site substrate with an amine nucleophile puromycin a 1 nucleophile hydroxypuromycin The relative rate ver sus pH profiles were indistinguishable although hy droxypuromycin reacted NZOOfold more slowly than puromycin both yielding an apparent pKa of 75 80 39 for 39 39 L 39 h quot r r xypuromycin is several units higher than for deprotonat ing the ammonium group on puromycin these data sug gested that the titrating group being observed was on the ribosome not the substrate This pKa was ascribed to either a histidine or an aamino group on one of the ribosomal proteins However as Knowles said the deductivejump from an apparent pKa of 65 to the state ment that histidine is in the active site is on the basis of pHdependence alone unwarranted and improper The case remained open An Atomic Wew of the Active Site Several decades passed with little progress made on the nature of the group with a nearneutral pKa involved Cell 666 my W Transllion Slate Analog in in the peptidyl transfer reaction until two years ago when Steitz Moore and colleagues determined an atomic resolution structure of the large subunit of the ribosome Ban et al 2000 By soaking into the crystals a previously characterized transition state analog l39 SA Figure 1 Welch et al 1995 they identi ed the site where peptide bond formation occurs Nissen et al 2000 Recent experiments have even demonstrated that ribosomes in these crystals can catalyze the formation of peptide bonds Schmeing et al 2002 This stunning view of the large ribosomal subunit demonstrated that ties directly involved in stabilizing the transition state for peptide bond formation must be nucleotide based While the nucleotides that were found in the active site were anticipated by years of biochemical and genetic experiments reviewed in Green and Noller 1997 knowledge of their organization is greatly empowering As in most active sites the nucleotides are tightly packed to form a cavity where the substrates bind and are oriented for catalysis Guanosines found in the A and P loops of the 238 rRNA directly interact with the CCA ends of the tRNA substrates by base pairing inter actions The regions of the ribosome nearest the transi tion state analog are composed exclusively of nucleo tides from the central loop of domain V of the 238 rRNA five nucleotides approach the phosphoramidate moiety of the TSA the analog of the tetrahedral center in thetransition state Figure1within 6 A Which if any of these nucleotides are directly involved in stabilizing the transition state for peptide bond formation Nucleotide A2451 39g r 39 in me after math of the publication of the 508 subunit structure because it makes the closest approach to the phos phoramidate center of the TSA Nissen et al 2000 Indeed the N3 position of this adenosine residue is nearly within hydrogen bonding distance 34 A of one of the nonbridging oxygen atoms of the TSA For such a close approach to be made it was proposed that a proton must be found there though neither the pKa in solution of the N3 of adenosine lt1 nor of the oxygen of the phosphoramide 31 would normally allow for INC Figure1 Proposed Chemical l N 0N m1 f on tel 1 b 1 a n mln R 4 a n 3 w 1m m4 m an Reaction Mechanism for Peptide Bond Formation Groups that must lose a proton are in red and those that must gain a proton are in blue The P and A site tRNAs are on the left and right respectively The transition state analog used to locate the active site of the 505 subunit of 1 the ribosome is boxed Positions corre sponding to the carbonyl oxygen and the nu cleophilic amine are indicated with aqua and red arrows respectively 7 n protonation at the pH at which the structural data were obtained 58 The existence of such a proton could be reconciled by a perturbed pKa in the ribosome resulting from the structural constraints of the active site as has recently been proposed for the hepatitis delta virus ribo zyme Nakano et al 2000 A nucleotide with a suffi ciently perturbed pKa in the active site would be an excellent candidate to function as a general acid or base and assist in the catalysis of peptide bond formation as discussed above Figure 1 The Crystals Are Active But the Active Site Is Flexible Initial discussions of an unusual pKa for A2451 were fueled by speculation that a buried phosphate on neigh boring nucleotide A2450 and a charge relay network mig I 1 SpKauderu 39 v rue Classical chemical modification approaches pioneered by Westheimer and colleagues Schmidt and West heimer 1971 to probe the pKas of active site residues in proteins were used by Muth and Strobel Muth et al 2000 to provide data supporting the proposal that A2451 in E coli ribosomes had a pKa of N75 at least 4 units higher than in solution However subsequent studies by Bayfield et al 2001 showed that the appar ent pH dependence of DM8 modification at A2451 was a once more changes apparently affect the pH dependence of DM8 modification of bases in the active site though whether the conformational changes themselves are promoted by changes in pH has yet to be determined What re mains striking about these results however is the idea that the active site can adopt multiple conformations understanding the relationship between these different structural states and their catalytic potential may tell us something important about the biology and chemistry of protein synthesis Minireview 667 Figure 2 View of the Active Site of the Ribosome Focusing on Nu cleotides Proximal to the Bound Transition State Analog A2451 is in green and the A2450C2063 base pair is in red The purple strand is the TSA with the tetrahedral center in black Note that the model of the SOSTSA complex PDB ID 1FFZ is unrefined The figure was made by Silke Dorner using the program Ribbons M Carson Kinetic Insights into Proton Movement Knowles review warned the reader of the dangers awaiting those who undertake pH dependence studies However he conceded that if a single elementary step in an enzymecatalyzed reaction can be cleanly isolated over the whole pH range of interest then the pKavalue of a group on which this step depends will be properly determined In a compelling recent report by Rodnina and colleagues Katunin et al 2002 the active site of the ribosomewas studied with this concession in mind The authors designed an assay to measure the rate constant for peptide bond formation kpep in a complex with a dipeptidyl tRNA in the P site and puromycin in the A site and used this assay to study the pH depen dence of the peptidyl transferase reaction The plot of logkpep versus pH has a slope of 15 Ge greater than 10 suggesting that the titration of more than one ioniz ing group was being measured The data were fit well with a model for a system with two ionizing groups one titrating at pH 69 and the other at pH 75 the singly protonated species Ge the group with pKa 75 still pro tonated retaining significant activity N100fold below that of the fully unprotonated species Thus two different protons need to be removed from the ribosome andor the substrate to achieve the maxi mal rate of peptide bond formation What groups are responsible for these two titration points397 The pKa of 69 was relatively straightfonivard to assign Titration of puromycin in solution revealed that its nucleophilic amine has a pKa of 69 The relevance of this proton in the peptidyl transferase reaction was confirmed when the authors substituted hydroxypuromycin as the ac beplUl quot r I of the reaction changed First the reaction rate is N200 fold slower with hydroxypuromycin than puromycin consistent with the reduced nucleophilicity of a hydroxyl group relative to an amine and with the notion that the step being measured in the assay is the chemical step of peptide bond formation Second while the reaction remains pH dependent it is no longer dependent on the deprotonation of a group at pH 69 Furthermore the slope of the logkpep versus pH plot is now 1 consistent with a single titrating group remaining These data indi cate that the identity of the group that titrates at pH 69 in the peptidyl transferase reaction is the ammonium form NHg of puromycin Logic dictates that the other titratable proton must reside on the ribosome In a tour de force experiment requiring deconvolution of the kinetics of a heteroge neous wildtype and mutant ribosome population the authors then asked whether changing the identity of the notorious nucleotide A2451 affected the pHrate profile of the peptidyl transferase reaction A2451 U mutant ri bosomes catalyze peptide bond formation 130 times more slowly than wildtype ribosomes and the pH de pendence of the reaction indeed now reflects the titra tionofunly 39 a39 r quot r39 nfsr 39 These results are in line with what would be predicted if A2451 acts as a general base in this reaction Sounds Great So What s the Problem In light of Knowles warnings it is worth thinking through u that by the ribosome before concluding that A2451 plays such a role First the fact that the reaction with hydroxy puromycin is dependent on a deprotonated group on the ribosome with a pKa of N75 suggests that if this group is acting as a general base it is involved in ab stracting a proton from the unprotonated form of the nucleophile the free amine NHZ in the case of puro mycin At the start of the reaction the pKa of this proton is gt30 and thus it is not possible that a base with a pKa of 75 could remove it As the reaction proceeds Figure 1 the pKa of this proton decreases and so a general base could be useful in the transition state Even then however the pKa of the most basic atom in an adenosine N1 would have to be shifted by four units or it to act as a general base at physiological pH To make matters worse in the crystal structure of the 508 subunit A2451 s N1 is pointed away from the transition state analog and is involved in a hydrogen bond with G2061 Nissen et al 2000 while its N3 points toward the TSA Finally the only evidence that the pKa of A2451 is actually perturbed is the close approach in the crystal structure of one of its electronegative groups N3 and the nonbridging oxygen of the phosphoramidate TSA Given that protons are not visible in the 508 structure at this level of resolution the possibility should be enter tained that these two unprotonated electronegative groups are able to make a very close approach in the TSAribosome complex because the binding energy provided by the rest of the TSA and the P and A loops of the 238 rRNA compensates for this negative interaction Parnell et al 2002 How else can we explain what looks at first glance to quot 39 39 39 dse ll 39 a o r 1 as l that the protonated form of A2451 acts as an oxyanion hole to stabilize the negative charge on the carbonyl oxygen in the transition state Nissen et al 2000 The fact that the rate of the reaction increases as the pH increases seems to run counter to what one would ex pect if A2451H acted to stabilize the oxyanion al though more complicated scenarios that would allow protonated A2451 to act as an oxyanion hole or general acid while still yielding the observed pHrate profile are possible Jencks 1969 Nakano et al 2000 To begin to address these issues Strobel and colleagues Parnell et al 2002 recently demonstrated that the affinity of the ribosome for the TSA is independent of pH between