Active Site Structural Changes Research Articles

Revealing substrate-induced structural changes in active site of human CYP51 in the presence of its physiological substrates

The human sterol 14α-demethylases (CYP51, CYP is an abbreviation for cytochrome P450) catalyze three-step oxidative removal of 14α-methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally conducting a CC bond cleavage reaction. This present study utilizes a combination of Resonance Raman spectroscopy and Nanodisc technology to probe the active site structure of CYP51 in the presence of its hydroxylase and lyase substrates. Ligand-binding induced partial low-to-high-spin conversion is observed by applying electronic absorption spectroscopy and Resonance Raman (RR) spectroscopy. This low degree of spin conversion of CYP51 is contributed by the retention of the water ligand coordinated to the heme iron as well as direct interaction between the hydroxyl group of lyase substrate and the iron center. No significant changes in active site structure are found between detergent-stabilized CYP51 and nanodisc-incorporated CYP51, nevertheless, it is demonstrated that nanodisc-incorporated assemblies provide much more well-defined active site RR spectroscopic responses, which induces a larger conversion from low-to-high-spin state in presence of the substrates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential CC bond cleavage reaction.

Importance of Asparagine 202 in Manipulating Active Site Structure and Substrate Preference for Human CYP17A1.

The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKB─P05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.

The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4

Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug–drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV–VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4.

Resonance Raman spectroscopy reveals pH-dependent active site structural changes of lactoperoxidase compound 0 and its ferryl heme O-O bond cleavage products.

The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this "stored" proton to the hydroperoxo fragment, it rapidly undergoes O-O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe-O-O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O-O bond-cleavage product, whose ν(Fe═O) frequencies reflect substantially different Fe═O bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe-O linkage of the Fe-O-O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe-O-O and Fe═O fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins.

Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5′-Phosphosulfate Kinase

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp(136), which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

Dependence of Catalytic Ability of Catalase‐peroxidase on Intersubunit Interactions

Catalase-peroxidases (KatGs) serve as an ideal model to explore the role of distant protein structures and their interactions on enzyme active site structure and catalysis. KatG efficiently facilitates hydrogen peroxide decomposition by two pathways: catalase and peroxidase. However, the KatG active site is nearly identical to those of classical heme peroxidases (e.g., cytochrome c peroxidase), which have poor catalase activity. Obvious differences between these two groups of enzymes include a gene duplicated C-terminal domain and two interhelical insertions unique to KatG. Deletion of any of these structures has deep impact on KatG active site environment and catalytic function (e.g., selective loss of catalase activity). To study the role of distant interactions between two of these KatG-unique features, site-directed and deletion mutagenesis were evaluated for changes in active site structure by various spectroscopies and steady state parameters of catalase and peroxidase activities. Strikingly, the deletion variants showed near to complete loss of catalase activity but retention of appreciable peroxidase activity. Concomitant with shift in activity was a change in heme coordination from predominantly hexacoordinate high-spin to predominantly pentacoordinate high-spin. These results emphasize the importance of distant protein structures and their interactions for fine-tuning active site function, and in addition, point to factors beyond simple sequence differences which control active site function even some 30 Å from the active site. This project was supported by National Science Foundation (MCB- 0641614).

Resonance Raman Studies of Cytochrome P450 2B4 in Its Interactions with Substrates and Redox Partners

Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.

Role of the C-Terminal Region of the B Component of Methylosinus trichosporium OB3b Methane Monooxygenase in the Regulation of Oxygen Activation

The effects of the C-terminal region of the B component (MMOB) of soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b on steady-state turnover, the transient kinetics of the reaction cycle, and the properties of the sMMO hydroxylase (MMOH) active site diiron cluster have been explored. MMOB is known to have many profound effects on the rate and specificity of sMMO. Past studies have revealed specific roles for the well-folded core structure of MMOB as well as the disordered N-terminal region. Here, it is shown that the disordered C-terminal region of MMOB also performs critical roles in the regulation of catalysis. Deletion mutants of MMOB missing 5, 8, and 13 C-terminal residues cause progressive decreases in the maximum steady-state turnover number, as well as lower apparent rate constants for formation of the key reaction cycle intermediate, compound Q. It is shown that this latter effect is actually due to a decrease in the rate constant for formation of an earlier intermediate, probably the hydroperoxo species, compound P. Moreover, the deletions result in substantial uncoupling at or before the P intermediate. It is proposed that this is due to competition between slow H(2)O(2) release from one of the intermediates and the reaction that carries this intermediate on to the next step in the cycle, which is slowed by the mutation. Electron paramagnetic resonance (EPR) studies of the hydroxylase component (MMOH) in the mixed valent state suggest that complexation with the mutant MMOBs alters the electronic properties of the diiron cluster in a manner distinct from that observed when wild-type MMOB is used. Active site structural changes are also suggested by a substantial decrease in the deuterium kinetic isotope effect for the reaction of Q with methane thought to be associated with a decrease in quantum tunneling in the C-H bond breaking reaction. Thus, the surface interactions between MMOH and MMOB that affect substrate oxidation and its regulation appear to require the complete MMOB C-terminal region for proper function.

Evolution of Enzymatic Activities in the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily: Enhancing the Promiscuous d-arabino-Hex-3-ulose 6-Phosphate Synthase Reaction Catalyzed by 3-Keto-l-gulonate 6-Phosphate Decarboxylase

3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction in the catabolic pathway of l-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J.Bacteriol. 184, 302-306]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.

Raman evidence that the lyoprotectant poly(ethylene glycol) does not restore nativity to the heme active site of horseradish peroxidase suspended in organic solvents.

Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.

Structural and Mechanistic Analysis of Two Refined Crystal Structures of the Pyridoxal Phosphate-dependent Enzyme Dialkylglycine Decarboxylase

Two refined structures, differing in alkali metal ion content, of the bifunctional, pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (DGD) are presented in detail. The enzyme is an α tetramer, built up as a dimer of dimers, with a subunit molecular mass of 46.5 kDa. The fold of DGD is similar to those of aspartate aminotransferase, ω-amino acid aminotransferease and tyrosine phenol-lyase. The structure has two binding sites for alkali metal ions. GDG with potassium in site 1 (near the active site) and sodium in site 2 (at the surface of the molecule) has been refined against 2.6Å resolution data ( R-factor=17.6%), and DGD with sodium at both sites has been refined against 2.1 Å resolution data ( R-factor=17.8%). The proximity of site 1 to the active site accounts for the dependence of enzyme activity on potassium ions, and the observed active site structural changes caused by ion exchange at this site explain the inhibition of activity by sodium. DGD catalyzes both the decarboxylation of dialkylglycine species and the transamination of l-amino acids in its normal catalytic cycle. The active site structure of DGD is moderately homologous to that of aspartate aminotransferase, which catalyzes only transamination; both the differences and similarities provide mechanistic guidelines for the DGD-catalyzed reactions. Models of the l-isovaline and l-alanine external aldimine intermediates suggest mechanisms by which the decarboxylation and transamination reactions could be accomplished within the single active site. Decarboxylatio is proposed to be at least partially catalyzed by stereo-electronic activation of the C α-carboxylate bond achieved by orienting this bond perpendicular to the plane of the pyridinium ring in the dialkylglycine external aldimine intermediate. Transamination is proposed to be catalyzed by a similar effect on the C α-H bond of the l-amino acid external aldimine intermediate, combined with general base catalysis provided by Lys272, in analogy to the mechanism of as partate aminotransferase.

Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

A series of derivatives for both the arthropod and mollusc hemocyanin biopolymers has been prepared; the derivatives contain a small fraction of electron paramagnetic resonance-detectable half-met [Cu(II) CU(I)] sites dispersed among the non-detectable oxy binuclear copper active sites. Upon deoxygenation, large changes in the electron paramagnetic resonance signal of these half-met spectral probe derivatives are observed, which are further adjusted by the heterotropic effectors Ca2+ and H+. The active site structural changes indicated that these spectral changes as the hemocyanins go from a relaxed to a tensed quaternary structure are then discussed.

Characterization of the Active Site Structures of Arginine Kinase-Substrate Complexes: WATER PROTON MAGNETIC RELAXATION RATES AND ELECTRON PARAMAGNETIC RESONANCE SPECTRA OF MANGANOUS-ENZYME COMPLEXES WITH SUBSTRATES AND OF A TRANSITION STATE ANALOG

Abstract The apparent number, n, of water ligands coordinated to bound Mn(II) in the ternary arginine kinase-MnADP or ATP complexes, in the abortive quaternary complex E-MnADP-arginine and in the transition state analog complex formed by addition of nitrate to the quaternary complex was estimated from the frequency dependence of the longitudinal proton relaxation rate of water (PRR) of lobster (Homarus americanus) muscle arginine kinase complexes. The apparent number of residual water ligands becomes progressively smaller as successive sites on the enzyme become occupied and is approximately 2 for the ternary nucleotide complexes, 1 for the abortive quaternary complex and considerably less than 1 for the transition state analog complex. The low values of n may be ascribed to the successive substitution of water ligands by protein ligands as each substrate and anion is successively bound to the enzyme or alternatively to an apparent rather than real disappearance of water ligands. The latter explanation implies that conformational changes induced by successive occupation of the binding sites on the enzyme are of such a nature that the rate of exchange of Mn(II)-bound water with solvent water becomes so slow for some Mn(II) water ligands that only a fraction of the total Mn(II)-bound water exchanges sufficiently rapidly to contribute to the PRR of bulk solvent. From the frequency dependence of the PRR of the ternary and quaternary complexes, it has been established that T1e, the electron spin relaxation time, is the dominant term in the correlation time which modulates the interaction between the Mn(II) electron spin and the water proton nuclear spin. Values of the electron spin relaxation times, T1e, estimated from PRR experiments are consistent with the lower limits of T1e obtained from line widths of the EPR spectra of the same complexes. No change is observed in the position of the peaks in the EPR spectra in proceeding from the binary MnADP complex to the ternary E-MnADP to the abortive quaternary E-MnADP-arginine or from MnATP to E-MnATP, and it may be inferred that there has been no gross modification in the symmetry of the electronic environment of Mn(II). Small changes in the line widths of the EPR spectra for these complexes do indicate that the accessibility of the Mn(II) liganded structure to collision with solvent molecules has been modified consistent with changes in the number of exchangeable water protons determined from PRR. Addition of nitrate to the abortive quaternary complex to form the transition state analog, as in the case of rabbit muscle creatine kinase, induces significant changes in the active site structure of arginine kinase as reflected in the altered EPR line shape. In this case altered line positions of the EPR spectrum also are observed. The latter indicates an asymmetry of the electronic environment of Mn(II) which may result from a substitution of ligands. The corresponding transition state analog for creatine kinase exhibits a much more anisotropic EPR spectrum. The EPR spectra are much more sensitive than PRR values to subtle changes in active site structure, e.g. spectral changes induced by d-arginine differ markedly from those produced by l-arginine in the presence of nitrate. Thiocyanate, nitrite, and formate at relatively high concentrations also modified the active site structure of arginine kinase, as evidenced by the alterations in the EPR spectrum of the quaternary complex upon addition of these anions. Modifications of the EPR spectra caused by these anions were qualitatively similar to those of nitrate but produced smaller changes in peak positions.