Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure.

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.

MALDI Peptide Mapping for Fast Analysis in Protein Footprinting.

Although protein footprinting results are commonly obtained by ESI-based LC-MS/MS, a more rapid-turnaround alternative approach is desirable to expand the scope of protein footprinting and facilitate routine analysis such as monitoring protein high order structure in quality control or checking epitope maps. Considering that MALDI is a faster procedure that can be easily adapted for high-throughput analysis, we explore here the feasibility of developing a MALDI-based analysis "portfolio" of bottom-up peptide mass mapping for footprinting. The approach was applied to several model proteins that were submitted to two footprinting strategies, FPOP and GEE labeling, and their performance was evaluated. We found adequate coverage that can be improved with automatic off-line separation and spotting, demonstrating the capability to footprint accurately protein conformational change, showing that MALDI may be useful for selected applications in protein footprinting.

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry.

Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.

A Single Approach Reveals the Composite Conformational Changes, Order of Binding, and Affinities for Calcium Binding to Calmodulin.

We found that a newly developed method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) can characterize section-by-section of a protein the conformational changes induced by metal-ion binding. Peptide-level LITPOMS applied to Ca2+ binding to calmodulin reveals binding order and site-specific affinity, providing new insights on the behavior of proteins upon binding Ca2+. We established that EF hand-4 (EF-4) binds calcium first, followed by EF-3, EF-2, and EF-1 and determined the four affinity constants by modeling the extent-of-modification curves. We also found positive cooperativity between EF-4, EF-3 and EF-2, EF-1 and allostery involving the four EF-hands. LITPOMS recapitulates via one approach the calcium-calmodulin binding that required decades of sophisticated development to afford versatility, comprehensiveness, and outstanding spatial resolution.

Composite Conformational Changes of Signaling Proteins upon Ligand Binding Revealed by a Single Approach: Calcium-Calmodulin Study.

Signaling proteins exemplified by calmodulin usually bind cooperatively to multiple ligands. Intermediate states and allosteric behavior are difficult to characterize. Here we extend a recently reported mass spectrometry (MS)-based method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) that characterizes complex binding systems typically found as signaling proteins. As reported previously, calmodulin's response to binding four Ca2+ can be determined by LITPOMS to reveal binding sites, binding order, and most importantly composite binding behavior. Modeling this behavior provides site-specific binding affinities. In this article, we dissect the composite, peptide-level conformational changes at several regions either by digestion with a different protease or by tandem MS of LITPOMS behavior at the amino-acid residue level. Such dissection greatly elevates spatial resolution and increases the confidence of binding-order assignment. These complementary views of complex protein conformational change recapitulate the cumulative understanding via a single approach, providing new insights on poorly understood yet important allostery and underpin an approach applicable for exploring other signaling systems.

Hydroxyl-Radical Reaction Pathways for the Fast Photochemical Oxidation of Proteins Platform As Revealed by 18O Isotopic Labeling.

Fast photochemical oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical (•OH) generated by photolysis of hydrogen peroxide (H2O2) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of •OH oxidative modification of 13 amino acid residues by using 18O isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H2O2. Methionine residues competitively take oxygen from H2O2 and dissolved oxygen (O2), whereas the remaining residues take oxygen exclusively from O2. Results reported in this work deepen the understanding of •OH labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology.

Fast photochemical oxidation of proteins (FPOP) is a footprinting technique used in mass spectrometry-based structural proteomics. It has been applied to solve a variety of problems in different areas of biology. A FPOP platform requires a laser, optics, and sample flow path properly assembled to enable fast footprinting. Sample preparation, buffer conditions, and reagent concentrations are essential to obtain reasonable oxidations on proteins. FPOP samples can be analyzed by LC-MS methods to measure the modification extent, which is a function of the solvent-accessible surface area of the protein. The platform can be expanded to accommodate several new approaches, including dose-response studies, new footprinting reagents, and two-laser pump-probe experiments. Here, we briefly review FPOP applications and in a detailed manner describe the procedures to set up an FPOP protein footprinting platform.

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen-Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3.

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 µM for the three different regions, giving an overall binding affinity of 7.4 µM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.

Understanding Curli Amyloid-Protein Aggregation by Hydrogen-Deuterium Exchange and Mass Spectrometry.

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect ß-strand repeating units (labeled R1-R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting.

To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.

Conformational-Sensitive Fast Photochemical Oxidation of Proteins and Mass Spectrometry Characterize Amyloid Beta 1-42 Aggregation.

Preventing and treating Alzheimer's disease require understanding the aggregation of amyloid beta 1-42 (Aß1-42) to give oligomers, protofibrils, and fibrils. Here we describe footprinting of Aß1-42 by hydroxyl radical-based fast photochemical oxidation of proteins (FPOP) and mass spectrometry (MS) to monitor the time-course of Aß1-42 aggregation. We resolved five distinct stages characterized by two sigmoidal behaviors, showing the time-dependent transitions of monomers-paranuclei-protofibrils-fibrillar aggregates. Kinetic modeling allows deciphering the amounts and interconversion of the dominant Aß1-42 species. Moreover, the irreversible footprinting probe provides insights into the kinetics of oligomerization and subsequent fibrillar growth by allowing the conformational changes of Aß1-42 at subregional and even amino-acid-residue levels to be revealed. The middle domain of Aß1-42 plays a major role in aggregation, whereas the N-terminus retains most of its solvent-accessibility during aggregation, and the hydrophobic C-terminus is involved to an intermediate extent. This approach affords an in situ, real-time monitoring of the solvent accessibility of Aß1-42 at various stages of oligomerization, and provides new insights on site-specific aggregation of Aß1-42 for a sample state beyond the capabilities of most other biophysical methods.

Pulsed hydrogen-deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aß) peptide aggregation.

Probing the conformational changes of amyloid beta (Aß) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aß42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aß42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aß42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu(2+) ions. This MS approach has sufficient structural resolution to allow interrogation of Aß aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.

Continuous and pulsed hydrogen-deuterium exchange and mass spectrometry characterize CsgE oligomerization.

We report the use of hydrogen-deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE.

Exploration of Resveratrol as a Potent Modulator of α-Synuclein Fibril Formation.

The molecular determinants of amyloid protein misfolding and aggregation are key for the development of therapeutic interventions in neurodegenerative disease. Although small synthetic molecules, bifunctional molecules, and natural products offer a potentially advantageous approach to therapeutics to remodel aggregation, their evaluation requires new platforms that are informed at the molecular level. To that end, we chose pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to discern the phenomena of aggregation modulation for a model system of alpha synuclein (αS) and resveratrol, an antiamyloid compound. We invoked, as a complement to HDX, advanced kinetic modeling described here to illuminate the details of aggregation and to determine the number of oligomeric populations by kinetically fitting the experimental data under conditions of limited proteolysis. The misfolding of αS is most evident within and nearby the nonamyloid-ß component region, and resveratrol significantly remodels that aggregation. HDX distinguishes readily a less solvent-accessible, more structured oligomer that coexists with a solvent-accessible, more disordered oligomer during aggregation. A view of the misfolding emerges from time-dependent changes in the fractional species across the protein with or without resveratrol, while details were determined through kinetic modeling of the protected species. A detailed picture of the inhibitory action of resveratrol with time and regional specificity emerges, a picture that can be obtained for other inhibitors and amyloid proteins. Moreover, the model reveals that new states of aggregation are sampled, providing new insights on amyloid formation. The findings were corroborated by circular dichroism and transmission electron microscopy.

Carboxyl-group footprinting maps the dimerization interface and phosphorylation-induced conformational changes of a membrane-associated tyrosine kinase.

Her4 is a transmembrane receptor tyrosine kinase belonging to the ErbB-EGFR family. It plays a vital role in the cardiovascular and nervous systems, and mutations in Her4 have been found in melanoma and lung cancer. The kinase domain of Her4 forms a dimer complex, called the asymmetric dimer, which results in kinase activation. Although a crystal structure of the Her4 asymmetric dimer is known, the dimer affinity and the effect of the subsequent phosphorylation steps on kinase domain conformation are unknown. We report here the use of carboxyl-group footprinting MS on a recombinant expressed, Her4 kinase-domain construct to address these questions. Carboxyl-group footprinting uses a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of glycine ethyl ester, to modify accessible carboxyl groups on glutamate and aspartate residues. Comparisons of Her4 kinase-domain monomers versus dimers and of unphosphorylated versus phosphorylated dimers were made to map the dimerization interface and to determine phosphorylation induced-conformational changes. We detected 37 glutamate and aspartate residues that were modified, and we quantified their extents of modification by liquid chromatography MS. Five residues showed changes in carboxyl-group modification. Three of these residues are at the predicted dimer interface, as shown by the crystal structure, and the remaining two residues are on loops that likely have altered conformation in the kinase dimer. Incubating the Her4 kinase dimers with ATP resulted in dramatic increase in Tyr-850 phosphorylation, located on the activation loop, and this resulted in a conformational change in this loop, as evidenced by reduction in carboxyl-group modification. The kinase monomer-dimer equilibrium was measured using a titration format in which the extent of carboxyl-group footprinting was mathematically modeled to give the dimer association constant (1.5-6.8 × 10(12) dm(2)/mol). This suggests that the kinase-domain makes a significant contribution to the overall dimerization affinity of the full-length Her4 protein.

Toward a MALDI in-source decay (ISD) method for top-down analysis of protein footprinting.

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.

Fast photochemical oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino-acid level.

We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast photochemical oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose "protection" changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix(1) shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix(1) formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range.

HD exchange and PLIMSTEX determine the affinities and order of binding of Ca2+ with troponin C.

Troponin C (TnC), present in all striated muscle, is the Ca(2+)-activated trigger that initiates myocyte contraction. The binding of Ca(2+) to TnC initiates a cascade of conformational changes involving the constituent proteins of the thin filament. The functional properties of TnC and its ability to bind Ca(2+) have significant regulatory influence on the contractile reaction of muscle. Changes in TnC may also correlate with cardiac and various other muscle-related diseases. We report here the implementation of the PLIMSTEX strategy (protein ligand interaction by mass spectrometry, titration, and H/D exchange) to elucidate the binding affinity of TnC with Ca(2+) and, more importantly, to determine the order of Ca(2+) binding of the four EF hands of the protein. The four equilibrium constants, K(1) = (5 ± 5) × 10(7) M(-1), K(2) = (1.8 ± 0.8) × 10(7) M(-1), K(3) = (4.2 ± 0.9) × 10(6) M(-1), and K(4) = (1.6 ± 0.6) × 10(6) M(-1), agree well with determinations by other methods and serve to increase our confidence in the PLIMSTEX approach. We determined the order of binding to the four EF hands to be III, IV, II, and I by extracting from the H/DX results the deuterium patterns for each EF hand for each state of the protein (apo through fully Ca(2+) bound). This approach, demonstrated for the first time, may be general for determining binding orders of metal ions and other ligands to proteins.

Interactions of apurinic/apyrimidinic endonuclease with a redox inhibitor: evidence for an alternate conformation of the enzyme.

Apurinic/apyrimidinic endonuclease (APE1) is an essential base excision repair protein that also functions as a reduction and oxidation (redox) factor in mammals. Through a thiol-based mechanism, APE1 reduces a number of important transcription factors, including AP-1, p53, NF-κB, and HIF-1α. What is known about the mechanism to date is that the buried residues Cys 65 and Cys 93 are critical for APE1's redox activity. To further detail the redox mechanism, we developed a chemical footprinting-mass spectrometric assay using N-ethylmaleimide (NEM), an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When APE1 was incubated with E3330, two NEM-modified products were observed, one with two and a second with seven added NEMs; this latter product corresponds to a fully modified APE1. In a similar control reaction without E3330, only the +2NEM product was observed in which the two solvent-accessible Cys residues, C99 and C138, were modified by NEM. Through hydrogen-deuterium amide exchange with analysis by mass spectrometry, we found that the +7NEM-modified species incorporates approximately 40 more deuterium atoms than the native protein, which exchanges nearly identically as the +2NEM product, suggesting that APE1 can be trapped in a partially unfolded state. E3330 was also found to increase the extent of disulfide bond formation involving redox critical Cys residues in APE1 as assessed by liquid chromatography and tandem mass spectrometry, suggesting a basis for its inhibitory effects on APE1's redox activity. Collectively, our results suggest that APE1 adopts a partially unfolded state, which we propose is the redox active form of the enzyme.