Substrate-induced Conformational Changes Research Articles

Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 Å to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.

Antlion Strategy for Enzyme Specificity

The role of protein conformational dynamics in enzyme specificity and efficiency has held the fascination of enzymologists since the original debates over “lock and key” versus “induced-fit” mechanisms. Recent data has shown that the large, substrate-induced conformational changes are an important governor of enzyme specificity. Molecular dynamics simulations hold the promise for atomically detailed analysis of induced-fit mechanisms, but the large spatial and temporal scales are a challenge. Therefore, conformational transitions and induced fits on the millisecond scale have been studied infrequently and represent a major frontier in computer simulation. Here we investigated induced-fit mechanisms using Directional Milestoning applied to HIV reverse transcriptase for both correct and mismatched base pairs. The predicted rate and free energy profiles agree with available experimental data, including new single molecule kinetic measurements. The substrate-induced conformational change proceeds through a transition-state with motions of up to 25 A in approximately 250 μs. The induced-fit mechanism affords specificity based upon a kinetic rather than a thermodynamic selection, which we liken to the manner in which an antlion captures its prey by digging a hole in the sand and waiting. Ants fall into the hole and are slow to escape and so they are eaten, whereas larger insects, which might see the antlion as prey, rapidly climb out of the hole. The substrate-induced conformational change is rapid and affords fast sampling of the bound nucleotide. A correct nucleotide leads to tight binding and alignment of catalytic residues to promote catalysis while a mismatched base precludes the formation of the tight binding state and the rapid opening of the specificity loop affords release of the mismatch.

Dirlotapide as a model substrate to refine structure-based drug design strategies on CYP3A4-catalyzed metabolism

Multiple crystal structures of CYP3A4 bound with various substrates or inhibitors have been used as templates for docking of new chemical entities to predict sites of metabolism and molecular interactions for drug design. Herein, modeling studies with dirlotapide, a CYP3A4 substrate, indicated that a substantial conformational change of CYP3A4 was necessary to accommodate it within the active site cavity, which is in good agreement with a new published CYP3A4 ritonavir co-crystal structure. Thus, the importance of considering the substrate-induced conformational change in CYP3A4, thermochemical properties of reaction centers, and essential in vitro experimental data support were analyzed for the refinement of computational models.

Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

Retuning Rieske-type Oxygenases to Expand Substrate Range

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

Role of Induced Fit in Limiting Discrimination against AZT by HIV Reverse Transcriptase

Single turnover kinetic studies were conducted using fluorescently labeled HIV reverse transcriptase (RT) to evaluate the role of nucleotide-induced changes in enzyme structure in the selectivity against AZT in order to explore why AZT-resistant forms of the enzyme fail to significantly discriminate against AZT. Fluorescent labeling of HIV RT provided a signal to monitor the isomerization from "open" to "closed" states following nucleotide binding. We measured the rate constants governing nucleotide binding and enzyme isomerization for TTP and AZT-triphosphate by the wild-type and AZT-resistant forms of the enzyme containing the thymidine analogue mutations (TAMs). We show that the TAMs alter the kinetics of AZT incorporation by weakening ground-state nucleotide binding and decreasing the rate of chemistry relative to the wild-type enzyme. However, the slower rate of incorporation of AZT by the TAMs HIV RT is counterbalanced a lower K(m), resulting from the equilibration of the conformational change step. In contrast, the K(m) for the wild-type enzyme reflects the balance between rates of binding and incorporation so the conformational change step does not come to equilibrium. These data once again demonstrate that the rate of substrate release, limited by the reverse of the substrate-induced conformational change, is the key determinant of the role of induced fit in enzyme specificity. Mutations leading to slower rates of incorporation have the unfortunate consequence of lowering the K(m) value by allowing the conformational change step to come to equilibrium.

PTMs in Conversation: Activity and Function of Deubiquitinating Enzymes Regulated via Post-Translational Modifications

Deubiquitinating enzymes (DUBs) constitute a diverse protein family and their impact on numerous biological and pathological processes has now been widely appreciated. Many DUB functions have to be tightly controlled within the cell, and this can be achieved in several ways, such as substrate-induced conformational changes, binding to adaptor proteins, proteolytic cleavage, and post-translational modifications (PTMs). This review is focused on the role of PTMs including monoubiquitination, sumoylation, acetylation, and phosphorylation as characterized and putative regulative factors of DUB function. Although this aspect of DUB functionality has not been yet thoroughly studied, PTMs represent a versatile and reversible method of controlling the role of DUBs in biological processes. In several cases PTMs might constitute a feedback mechanism insuring proper functioning of the ubiquitin proteasome system and other DUB-related pathways.

Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.

Arginine Kinase: Joint Crystallographic and NMR RDC Analyses Link Substrate-Associated Motions to Intrinsic Flexibility

The phosphagen kinase family, including creatine and arginine kinases (AKs), catalyzes the reversible transfer of a “high-energy” phosphate between ATP and a phosphoguanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of AK structures were interpreted as a plastic deformation. Here, the structure of Limulus substrate-free AK is refined against high-resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa) and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.7 Å transition-state analog complex shows large substrate-induced domain motions that can be broken down into movements of smaller quasi-rigid bodies. The solution-state structure of substrate-free AK is most consistent with an equilibrium of substrate-free and substrate-bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme and likely involve some degree of conformational selection.

Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase

CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate the mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503 V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. POR A287P and R457H had low activity with all substrates; Q153R had 76-94% of wild-type (WT) activity with midazolam and erythromycin, but 129-150% activity with testosterone and quinidine. The A503 V polymorphism reduced the CYP3A4 activity to 61-77% of WT with testosterone and midazolam, but had nearly WT activity with quinidine and erythromycin. POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably because of substrate-induced conformational changes in CYP3A4.

Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.

Using a homology model of cytochrome P450 2D6 to predict substrate site of metabolism

CYP2D6 is an important enzyme that is involved in first pass metabolism and is responsible for metabolizing ~25% of currently marketed drugs. A homology model of CYP2D6 was built using X-ray structures of ligand-bound CYP2C5 complexes as templates. This homology model was used in docking studies to rationalize and predict the site of metabolism of known CYP2D6 substrates. While the homology model was generally found to be in good agreement with the recently solved apo (ligand-free) X-ray structure of CYP2D6, significant differences between the structures were observed in the B' and F-G helical region. These structural differences are similar to those observed between ligand-free and ligand-bound structures of other CYPs and suggest that these conformational changes result from induced-fit adaptations upon ligand binding. By docking to the homology model using Glide, it was possible to identify the correct site of metabolism for a set of 16 CYP2D6 substrates 85% of the time when the 5 top scoring poses were examined. On the other hand, docking to the apo CYP2D6 X-ray structure led to a loss in accuracy in predicting the sites of metabolism for many of the CYP2D6 substrates considered in this study. These results demonstrate the importance of describing substrate-induced conformational changes that occur upon binding. The best results were obtained using Glide SP with van der Waals scaling set to 0.8 for both the receptor and ligand atoms. A discussion of putative binding modes that explain the distribution of metabolic sites for substrates, as well as a relationship between the number of metabolic sites and substrate size, are also presented. In addition, analysis of these binding modes enabled us to rationalize the typical hydroxylation and O-demethylation reactions catalyzed by CYP2D6 as well as the less common N-dealkylation.

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.

Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR

The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl(-) permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent (19)F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl(-) permeation pathway and show the usefulness of (19)F NMR for studying conformational changes in membrane proteins of known structure.

Substrate interactions of the electroneutral Na+‐coupled inorganic phosphate cotransporter (NaPi‐IIc)

The SLC34 solute carrier family comprises the electrogenic NaPi-IIa/b and the electroneutral NaPi-IIc, which display Na(+) : P(i) cotransport stoichiometries of 3 : 1 and 2 : 1, respectively. We previously proposed that NaPi-IIc lacks one of the three Na(+) interaction sites hypothesised for the electrogenic isoforms, but, unlike NaPi-IIa/b, its substrate binding order is undetermined. By expressing NaPi-IIc in Xenopus oocytes, isotope influx and efflux assays gave results consistent with Na(+) being the first and last substrate to bind. To further investigate substrate interactions, we applied a fluorometry-based technique that uses site-specific labelling with a fluorophore to characterize substrate-induced conformational changes. A novel Cys was introduced in the third extracellular loop of NaPi-IIc that could be labelled with a reporter fluorophore (MTS-TAMRA). Although labelling resulted in suppression of cotransport as previously reported for the electrogenic isoforms, changes in fluorescence were induced by changes in extracellular Na(+) concentration in the absence of P(i) and by changes in extracellular P(i) concentration in presence of Na(+). These data, combined with (32)P uptake data, also support a binding scheme in which Na(+) is the first substrate to interact. Moreover, the apparent P(i) affinity from fluorometry agreed with that from (32)P uptake, confirming the applicability of the fluorometric technique for kinetic studies of electroneutral carriers. Analysis of the fluorescence data showed that like the electrogenic NaPi-IIb, 2 Na(+) ions interact cooperatively with NaPi-IIc before P(i) binding, which implies that only one of these is translocated. This result provides compelling evidence that SLC34 proteins share common motifs for substrate interaction and that cotransport and substrate binding stoichiometries are not necessarily equivalent.

Substrate-induced Conformational Changes in the Essential Peripheral Membrane-associated Mannosyltransferase PimA from Mycobacteria: IMPLICATIONS FOR CATALYSIS

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the beta-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic alpha-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Triphosphate tunnel metalloenzymes (TTMs) are a superfamily of phosphotransferases with a distinctive active site located within an eight-stranded beta barrel. The best understood family members are the eukaryal RNA triphosphatases, which catalyze the initial step in mRNA capping. The RNA triphosphatases characteristically hydrolyze nucleoside 5'-triphosphates in the presence of manganese and are inept at cleaving inorganic tripolyphosphate. We recently identified a TTM protein from the bacterium Clostridium thermocellum (CthTTM) with the opposite substrate preference. Here we report that CthTTM catalyzes hydrolysis of guanosine 5'-tetraphosphate to yield GTP and P(i) (K(m) = 70 microm, k(cat) = 170 s(-1)) much more effectively than it converts GTP to GDP and P(i) (K(m) = 70 microm, k(cat) = 0.3 s(-1)), implying that a nucleoside interferes when positioned too close to the tunnel entrance. CthTTM is capable of quantitatively cleaving diadenosine hexaphosphate but has feeble activity with shorter derivatives diadenosine tetraphosphate and diadenosine pentaphosphate. We propose that the tunnel opens to accommodate the dumbbell-shaped diadenosine hexaphosphate and then closes around it to perform catalysis. We find that CthTTM can exhaustively hydrolyze a long-chain inorganic polyphosphate, a molecule that plays important roles in bacterial physiology. CthTTM differs from other known polyphosphatases in that it yields a approximately 2:1 mixture of P(i) and PP(i) end products. Bacterial/archaeal TTMs have a C-terminal helix located near the tunnel entrance. Deletion of this helix from CthTTM exerts pleiotropic effects. (i) It suppresses hydrolysis of guanosine 5'-tetraphosphate and inorganic PPP(i); (ii) it stimulates NTP hydrolysis; and (iii) it biases the outcome of the long-chain polyphosphatase reaction more strongly in favor of P(i) production. We discuss models for substrate binding in the triphosphate tunnel.

Conformational Events during Ternary Enzyme−Substrate Complex Formation Are Rate Limiting in the Catalytic Cycle of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

The light-driven enzyme, protochlorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower substrate binding events leading to the formation of the catalytically active ternary complex. As POR is light-activated, it is relatively straightforward to isolate the ternary enzyme-substrate complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are complex, involving multiple conformational states en route to the formation of a ternary complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the enzyme active site. This is followed by the binding of the Pchlide substrate and subsequent substrate-induced conformational changes within the enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein.

Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.