The role of nicotinamide adenine dinucleotide in the inhibition of bovine liver S-adenosylhomocysteine hydrolase by neplanocin A

Neoplanocin A, a cyclopentenyl analog of adenosine, has been shown recently to be a tight binding inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), exhibiting a stoichiometry of one molecule of inhibitor per molecule of the enzyme tetramer (Borchardt, R. T., Keller, B. T., and Patel-Thombre, U. (1984) J. Biol. Chem. 259, 4353-4358). In the present study a detailed analysis was performed of the possible role of the enzyme-bound NAD+ in the inactivation of AdoHcy hydrolase by neplanocin A. The NAD+/NADH content was quantitated using a fluorescence technique. The native enzyme showed intrinsic fluorescence with an emission maximum at 460 nm when excited at 340 nm, partially due to NADH bound to the enzyme. It was found that the content of NAD+ and NADH in freshly prepared, native enzyme is equal, having a stoichiometry of two nucleotides per enzyme molecule (tetramer). In addition, it was observed that the enzymatic activity of the native enzyme can be increased by about 30% following preincubation with NAD+. Furthermore, it was demonstrated that the mechanism of inhibition of AdoHcy hydrolase by neplanocin A involves the reduction of enzymatically bound NAD+ to NADH. Catalytic activity of the inactivated enzyme could be fully recovered in a time-dependent manner by further incubation with NAD+ (but not NADH). It was also found that inhibition by neplanocin A does not involve dissociation of the bound NAD+ or NADH from the enzyme, but simply reduction of the NAD+ to NADH.     

The mechanism of inhibition of Alcaligenes faecalis S-adenosylhomocysteine hydrolase by neplanocin A

Neplanocin A, a cyclopentenyl analog of adenosine, has been reported by S. Yaginuma, N. Muto, M. Tsujino, Y. Sudate, M. Hayashi, and M. Otari (1981) J. Antibiot. 34, 359-366 to exhibit antibacterial activity against Alcaligenes faecalis. Since neplanocin A (NpcA) is a known inhibitor of eukaryotic S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) (R. T. Borchardt, B. T. Keller, and U. Patel-Thombre (1984) J. Biol. Chem. 259, 4353-4358), the present study was undertaken to determine the effects of this carbocyclic nucleoside on AdoHcy hydrolase isolated from a prokaryotic source (A. faecalis). AdoHcy hydrolase was purified to homogeneity by affinity chromatography on an AdoHcy-agarose matrix from A. faecalis. Neplanocin A inactivated the purified AdoHcy hydrolase in a time- and concentration-dependent manner and the enzyme activity could not be recovered by dialysis. The inactivation of this bacterial enzyme by neplanocin A is accompanied by a reduction of three of the six enzyme-bound NAD+s to NADHs. These results suggest that the prokaryotic enzyme, like the eukaryotic AdoHcy hydrolase, is susceptible to inhibition by neplanocin A. The mechanism of inactivation in both cases appears to be a Kcat mechanism involving the reduction of the enzyme-bound NAD+ to NADH. The fact that total inhibition of the prokaryotic AdoHcy hydrolase by NpcA results in a reduction of only three of the six enzyme-bound NAD+s to NADHs suggests that the enzyme shows half-site reactivity (i.e., only three of the six subunits are catalytically active).     

Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolase

S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.     

Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.     

Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.     

Partially folded conformations of bovine liver glutamate dehydrogenase induced by mild acidic conditions

The acid-induced unfolding of bovine liver glutamate dehydrogenase (GDH) was studied using various spectroscopic methods such as far- and near-UV circular dichroism (CD), intrinsic and 1-anilino naphthalene-8-sulphonate (ANS) extrinsic fluorescence spectroscopy, light scattering and fluorescence quenching in 20 mM mixed buffer at various pHs. CD spectra show that at pH 3.5, GDH retains its secondary structure substantially, whereas its tertiary structure content is reduced considerably. Intrinsic fluorescence of GDH and ANS binding suggest that, at pH 3.5, the hydrophobic surface of enzyme is more exposed in comparison to the native form. Acrylamide quenching indicates more exposure of tryptophan residues of enzyme at pH 3.5 in comparison to pH 7.5. Another partially unfolded intermediate was detected at pH 5.0, which with its ANS binding capacity lies between the pH 3.5 intermediate and the native form of the enzyme. Gel filtration results revealed that the enzyme at pH 3.5 is dissociated into trimeric species whereas it exists as hexamer at pH 7.5 and 5.0. All the data taken together suggest the existence of two partially unfolded states of GDH at moderate acidic pHs which may be considered as molten and pre-molten globule-like states.     

Inactivation of S-adenosylhomocysteine hydrolase with haloethyl and dihalocyclopropyl esters derived from homoadenosine-6'-carboxylic acid

In a search for new inhibitors that exploit 5'-6' 'hydrolytic activity' of AdoHcy hydrolase, a new series of haloethyl and dihalocyclopropyl esters 2-3 were designed and their interaction with the enzyme studied. Incubation of the enzyme with 2-3 resulted in time- and concentration-dependent inactivation of AdoHcy hydrolase as well as almost total depletion of its NAD(+) content. Further results indicated that the 'oxidative' but not the 'hydrolytic' activity was involved in the inactivation process.     

Inactivation of human S-adenosylhomocysteine hydrolase by covalent labeling of cysteine 195 with thionucleoside derivatives

A new series of 5'-thioadenosine derivatives 1-4 were synthesized for selectively targeting (195)Cys of human AdoHcy hydrolase. Their incubation with the enzyme resulted in time- and concentration-dependent inactivation, without major modifications of the NAD(+)/NADH ratio. The electrospray mass analysis of the inactivated enzyme with 1, 2, 3, and 4b showed that inhibition was accompanied by the formation of a specific and covalent labeling of each AdoHcy hydrolase subunit. Proteolytic cleavage (endo-Lys-C) and subsequent peptide characterization of the labeled enzyme revealed that (195)Cys was the residue modified during the inactivation process.     

Metastability of Papain and the Molecular Mechanism for its Sequential Acid-Denaturation

Acid unfolding of non-inhibited papain at pH 2 was studied by means of spectroscopic and electrophoresis techniques as well as activity assays. We found a molten globule like species (A state) similar to that previously reported for bromelain and S-carboxy-methyl-papain. We demonstrated that this A state is not thermodynamically stable but a metastable conformer which decays into an unfolded conformation in a few hours. The mechanism of acid unfolding to the A state proved to be completely irreversible, with a biphasic time evolution of spectroscopic signals characteristic of the existence of a kinetic intermediate. This latter species showed properties in-between native and A state such as secondary structure, exposition of hydrophobic area and tryptophan environment, but a native like hydrodynamic radius. Native papain seems to unfold at acid pH through at least two kinetic barriers, being its proregion mandatory to conduct and stabilize its active structure. Computer simulations of acid unfolding, followed by ANS docking, identified three regions of cavity formation induced by acid media which might be used as regions to be fortified by protein engineering in the quest for extreme-resistant proteases or as hot-spots for protease inactivation.     

Native hydrogen bonds in a molten globule: the apoflavodoxin thermal intermediate

The structure and energetics of protein-folding intermediates are poorly understood. We have identified, in the thermal unfolding of the apoflavodoxin from Anabaena PCC 7119, an equilibrium intermediate with spectroscopic properties of a molten globule and substantial enthalpy and heat capacity of unfolding. The structure of the intermediate is probed by mutagenesis (and phi analysis) of polar residues involved in surface-exposed hydrogen bonds connecting secondary-structure elements in the native protein. All hydrogen bonds analysed are formed in the molten globule intermediate, either with native strength or debilitated. This suggests the overall intermediate's topology and surface tertiary interactions are close to native, and indicates that hydrogen bonding may contribute significantly to shape the conformation and energetics of folding intermediates.     

Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH

A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS), hexa decyltrimethyl ammonium bromide (CTAB) and Tween-20] on the structure of acid-unfolded papain (EC.3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits a substantial amount of secondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydrochloride) but loses the persistent tertiary contacts of the native state. Addition of detergents caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Near-UV CD spectra also showed the regain of native-like spectral features in the presence of 8 mM SDS and 3.5 mM CTAB. Induction of structure in acid-unfolded papain was greater in the presence SDS followed by CTAB and Tween-20. Intrinsic tryptophan fluorescence studies indicate the change in the environment of tryptophan residues upon addition of detergents to acid-unfolded papain. Addition of 8 mM SDS resulted in the loss of ANS binding sites exhibited by a decrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. Maximum ANS binding was obtained in the presence of 0.1 mM Tween-20 followed by CTAB, indicating a compact "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. These results suggest that papain at low pH and in the presence of SDS exists in a partially folded state characterized by native-like secondary structure and tertiary folds. While in the presence of Tween, acid-unfolded papain exists as a compact intermediate with molten-globule-like characteristics, viz. enhanced hydrophobic surface area and retention of secondary structure. While in the presence of CTAB it exists as a compact intermediate with regain of native-like secondary and partial tertiary structure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a molten globule state with tertiary folds.     

Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.     

Single disulfide bond reduced papain exists in a compact intermediate state

Partially reduced proteins and other chemically modified derivatives are very useful model systems to understand the protein folding in vivo. Upon reduction, proteins attain different conformations with varying degrees of compactness. The reduction of papain in the presence of 8 M urea leads to the partial reduction of one disulfide bond. This derivative (single disulfide reduced carboxymethylated 1RCM papain (3RCM papain)) was characterized by spectroscopic methods and the effect of this reduction on the unfolding of the protein was investigated. Under this partial reduction, papain exhibits more than half of the tertiary and most of the secondary structures relative to the non-reduced molecule (free cysteine reduced and carboxymethylated papain (1RCM papain)). Hydrophobic regions are exposed to the solvent as observed through 8-anilino-1-naphthalene sulfonic acid binding which was absent in the fully intact and unfolded protein, at neutral pH. Hydrodynamic studies indicated that 3RCM papain, under neutral conditions, possess expanded conformation as compared to the native protein. Tryptophan fluorescence quenching studies suggested the exposure of aromatic residues to solvent. Guanidine hydrochloride induced unfolding of this derivative, at neutral pH, showed a non-cooperative transition contrary to the cooperativity seen with intact protein. Thermal unfolding indicates that 3RCM papain is less stable compared to the intact protein. These findings suggest that partial reduction of papain has a significant effect on the unfolding behavior of papain.     

Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates

The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis. The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities. A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding. The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu). Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding. Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone. The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.     

Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: Interaction with cofactors and effect of osmolytes on unfolding

Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.     

Chemical modification of tryptophan residues of leucyl tRNA synthetase by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide

The structural accessibility of tryptophan residues in leucyl-tRNA synthetase from cow mammary gland has been studied using chemical modifications by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. The modifications were monitored by UV absorbance and intrinsic fluorescence of the enzyme's tryptophan residues. Under native conditions, at pH 7,8, only two exposed tryptophan residues are modified in each subunit of the dimeric enzyme. Under denaturing conditions, in 6 M guanidine hydrochloride solution, internal tryptophan residues are also modified as a consequence of unfolding of the native tertiary structure of the enzyme. Modifications of tryptophan residues resulted in inactivation of leucyl-tRNA synthetase both in aminoacylation and ATP-PPi exchange reactions. In the specific complex of leucyl-tRNA synthetase with the cognate tRNALeu one of exposed tryptophan residues is protected by tRNALeu and is not modified by the above reagents.     

Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.     

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.