Effect of (R)-2-Amino-6-(1R,2S)-1,2-Dihydroxypropyl)-5,6,7,8-Tetrahydropterin-4(3H)-One and Its Structural Analogues on the Temperature Stability of Tryptophan Hydroxylase 2 with the P447R Mutation.

The enzyme tryptophan hydroxylase 2 (TPH2) catalyzes the hydroxylation of L-tryptophan to L-5-hydroxytryptophan (5-HTP), the first and the key step in 5-HT synthesis in the mammalian brain. Mutations in the human Tph2 gene reducing enzyme activity increase the risk of psychopathology. Pharmacological chaperones are small molecules that can specifically bind to mutant protein molecules, restore their disturbed 3D structure to the native state, and increase their stability and functional activity. The chaperone activity of (R)-2-amino-6-(1R,2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is expressed by increasing the in vitro thermal stability of mutant tyrosine hydroxylase and phenylalanine hydroxylase molecules which are similar to TPH2 in their structure and characteristics. The P447R substitution in the mouse TPH2 molecule results in a 2-fold decrease in enzyme activity in their brains. We studied the effect of this mutation on the TPH2 thermal stability, as well as on the ability of BH4 and its 8 structural analogues to increase the thermal stability of the mutant TPH2 from midbrain extracts of BALB/C mice. Temperature stability was studied by the decrease in enzyme activity during its heating for 2 min at increasing temperatures and was evaluated by the T50 value that is the temperature at which the enzyme activity decreased by half. For the mutant TPH2, the T50 value was decreased compared to the wild type enzyme. BH4 and its closest structural analogue, 6-methyl-5,6,7,8-tetrahydropterin, increased the T50 value, i.e., exhibited chaperone activity. Other close BH4 analogs, 6,7-dimethyl-5,6,7,8-tetrahydropterin and folic acid, were not effective. It can be assumed that BH4 can be effective in the treatment of mental disorders caused by mutations in the Tph2 gene.

Identification of Orthosteric and Allosteric Pharmacological Chaperones for Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.

Transcriptomic analysis identifies dysregulated pathways and therapeutic targets in PMM2-CDG.

PMM2-CDG (MIM # 212065), the most common congenital disorder of glycosylation, is caused by the deficiency of phosphomannomutase 2 (PMM2). It is a multisystemic disease of variable severity that particularly affects the nervous system; however, its molecular pathophysiology remains poorly understood. Currently, there is no effective treatment. We performed an RNA-seq based transcriptomic study using patient-derived fibroblasts to gain insight into the mechanisms underlying the clinical symptomatology and to identify druggable targets. Systems biology methods were used to identify cellular pathways potentially affected by PMM2 deficiency, including Senescence, Bone regulation, Cell adhesion and Extracellular Matrix (ECM) and Response to cytokines. Functional validation assays using patients' fibroblasts revealed defects related to cell proliferation, cell cycle, the composition of the ECM and cell migration, and showed a potential role of the inflammatory response in the pathophysiology of the disease. Furthermore, treatment with a previously described pharmacological chaperone reverted the differential expression of some of the dysregulated genes. The results presented from transcriptomic data might serve as a platform for identifying therapeutic targets for PMM2-CDG, as well as for monitoring the effectiveness of therapeutic strategies, including pharmacological candidates and mannose-1-P, drug repurposing.

Exploring the efficacy and safety of Ambroxol in Gaucher disease: an overview of clinical studies.

Gaucher disease (GD) is mainly caused by glucocerebrosidase (GCase) enzyme deficiency due to genetic variations in the GBA1 gene leading to the toxic accumulation of sphingolipids in various organs, which causes symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, and neurological manifestations. GD is clinically classified into the non-neuronopathic type 1, and the acute and chronic neuronopathic forms, types 2 and 3, respectively. In addition to the current approved GD medications, the repurposing of Ambroxol (ABX) has emerged as a prospective enzyme enhancement therapy option showing its potential to enhance mutated GCase activity and reduce glucosylceramide accumulation in GD-affected tissues of different GBA1 genotypes. The variability in response to ABX varies across different variants, highlighting the diversity in patients' therapeutic outcomes. Its oral availability and safety profile make it an attractive option, particularly for patients with neurological manifestations. Clinical trials are essential to explore further ABX's potential as a therapeutic medication for GD to encourage pharmaceutical companies' investment in its development. This review highlights the potential of ABX as a pharmacological chaperone therapy for GD and stresses the importance of addressing response variability in clinical studies to improve the management of this rare and complex disorder.

Tetrahydrobiopterin (BH4) treatment stabilizes tyrosine hydroxylase: Rescue of tyrosine hydroxylase deficiency phenotypes in human neurons and in a knock-in mouse model.

Proteostatic regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, is crucial for maintaining proper brain neurotransmitter homeostasis. Variants of the TH gene are associated with tyrosine hydroxylase deficiency (THD), a rare disorder with a wide phenotypic spectrum and variable response to treatment, which affects protein stability and may lead to accelerated degradation, loss of TH function and catecholamine deficiency. In this study, we investigated the effects of the TH cofactor tetrahydrobiopterin (BH4) on the stability of TH in isolated protein and in DAn- differentiated from iPSCs from a human healthy subject, as well as from THD patients with the R233H variant in homozygosity (THDA) and R328W and T399M variants in heterozygosity (THDB). We report an increase in TH and dopamine levels, and an increase in the number of TH+ cells in control and THDA cells. To translate this in vitro effect, we treated with BH4 a knock-in THD mouse model with Th variant corresponding to R233H in patients. Importantly, treatment with BH4 significantly improved motor function in these mice, as demonstrated by increased latency on the rotarod test and improved horizontal activity (catalepsy). In conclusion, our study demonstrates the stabilizing effects of BH4 on TH protein levels and function in THD neurons and mice, rescuing disease phenotypes and improving motor outcomes. These findings highlight the therapeutic potential of BH4 as a treatment option for THDA patients with specific variants and provide insights into the modulation of TH stability and its implications for THD management.

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction.

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the ß-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on ß-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

pH-Responsive Trihydroxylated Piperidines Rescue The Glucocerebrosidase Activity in Human Fibroblasts Bearing The Neuronopathic Gaucher-Related L444P/L444P Mutations in GBA1 Gene.

Engineering bioactive iminosugars with pH-responsive groups is an emerging approach to develop pharmacological chaperones (PCs) able to improve lysosomal trafficking and enzymatic activity rescue of mutated enzymes. The use of inexpensive l-malic acid allowed introduction of orthoester units into the lipophilic chain of an enantiomerically pure iminosugar affording only two diastereoisomers contrary to previous related studies. The iminosugar was prepared stereoselectively from the chiral pool (d-mannose) and chosen as the lead bioactive compound, to develop novel candidates for restoring the lysosomal enzyme glucocerebrosidase (GCase) activity. The stability of orthoester-appended iminosugars was studied by 1 H NMR spectroscopy both in neutral and acidic environments, and the loss of inhibitory activity with time in acid medium was demonstrated on cell lysates. Moreover, the ability to rescue GCase activity in the lysosomes as the result of a chaperoning effect was explored. A remarkable pharmacological chaperone activity was measured in fibroblasts hosting the homozygous L444P/L444P mutation, a cell line resistant to most PCs, besides the more commonly responding N370S mutation.

Phosphorus Dendrimers for Metal-Free Ligation: Design of Multivalent Pharmacological Chaperones against Gaucher Disease.

The first phosphorus dendrimers built on a cyclotriphosphazene core and decorated with six or twelve monofluorocyclooctyne units were prepared. A simple stirring allowed the grafting of N-hexyl deoxynojirimycin inhitopes onto their surface by copper-free strain promoted alkyne-azide cycloaddition click reaction. The synthesized iminosugars clusters were tested as multivalent inhibitors of the biologically relevant enzymes ß-glucocerebrosidase and acid α-glucosidase, involved in Gaucher and Pompe lysosomal storage diseases, respectively. For both enzymes, all the multivalent compounds were more potent than the reference N-hexyl deoxynojirimycin. Remarkably, the final dodecavalent compound proved to be one of the best ß-glucocerebrosidase inhibitors described to date. These cyclotriphosphazene-based deoxynojirimycin dendrimers were then evaluated as pharmacological chaperones against Gaucher disease. Not only did these multivalent constructs cross the cell membranes but they were also able to increase ß-glucocerebrosidase activity in Gaucher cells. Notably, dodecavalent compound allowed a 1.4-fold enzyme activity enhancement at a concentration as low as 100 nM. These new monofluorocyclooctyne-presenting dendrimers may further find numerous applications in the synthesis of multivalent objects for biological and pharmacological purposes.

Potential Binding Sites of Pharmacological Chaperone NCGC00241607 on Mutant ß-Glucocerebrosidase and Its Efficacy on Patient-Derived Cell Cultures in Gaucher and Parkinson's Disease.

Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), cause Gaucher disease (GD) and are the most common genetic risk factor for Parkinson's disease (PD). Pharmacological chaperones (PCs) are being developed as an alternative treatment approach for GD and PD. To date, NCGC00241607 (NCGC607) is one of the most promising PCs. Using molecular docking and molecular dynamics simulation we identified and characterized six allosteric binding sites on the GCase surface suitable for PCs. Two sites were energetically more preferable for NCGC607 and located nearby to the active site of the enzyme. We evaluated the effects of NCGC607 treatment on GCase activity and protein levels, glycolipids concentration in cultured macrophages from GD (n = 9) and GBA-PD (n = 5) patients as well as in induced human pluripotent stem cells (iPSC)-derived dopaminergic (DA) neurons from GBA-PD patient. The results showed that NCGC607 treatment increased GCase activity (by 1.3-fold) and protein levels (by 1.5-fold), decreased glycolipids concentration (by 4.0-fold) in cultured macrophages derived from GD patients and also enhanced GCase activity (by 1.5-fold) in cultured macrophages derived from GBA-PD patients with N370S mutation (p < 0.05). In iPSC-derived DA neurons from GBA-PD patients with N370S mutation NCGC607 treatment increased GCase activity and protein levels by 1.1-fold and 1.7-fold (p < 0.05). Thus, our results showed that NCGC607 could bind to allosteric sites on the GCase surface and confirmed its efficacy on cultured macrophages from GD and GBA-PD patients as well as on iPSC-derived DA neurons from GBA-PD patients.

Pharmacological Chaperones and Protein Conformational Diseases: Approaches of Computational Structural Biology.

Whenever a protein fails to fold into its native structure, a profound detrimental effect is likely to occur, and a disease is often developed. Protein conformational disorders arise when proteins adopt abnormal conformations due to a pathological gene variant that turns into gain/loss of function or improper localization/degradation. Pharmacological chaperones are small molecules restoring the correct folding of a protein suitable for treating conformational diseases. Small molecules like these bind poorly folded proteins similarly to physiological chaperones, bridging non-covalent interactions (hydrogen bonds, electrostatic interactions, and van der Waals contacts) loosened or lost due to mutations. Pharmacological chaperone development involves, among other things, structural biology investigation of the target protein and its misfolding and refolding. Such research can take advantage of computational methods at many stages. Here, we present an up-to-date review of the computational structural biology tools and approaches regarding protein stability evaluation, binding pocket discovery and druggability, drug repurposing, and virtual ligand screening. The tools are presented as organized in an ideal workflow oriented at pharmacological chaperones' rational design, also with the treatment of rare diseases in mind.

Light-Triggered Control of Glucocerebrosidase Inhibitors: Towards Photoswitchable Pharmacological Chaperones.

Piperidine-based photoswitchable derivatives have been developed as putative pharmacological chaperones for glucocerebrosidase (GCase), the defective enzyme in Gaucher disease (GD). The structure-activity study revealed that both the iminosugar and the light-sensitive azobenzene are essential features to exert inhibitory activity towards human GCase and a system with the correct inhibition trend (IC50 of the light-activated form lower than IC50 of the dark form) was identified. Kinetic analyses showed that all compounds are non-competitive inhibitors (mixed or pure) of GCase and the enzyme allosteric site involved in the interaction was identified by means of MD simulations. A moderate activity enhancement of mutant GCase assessed in GD patients' fibroblasts (ex vivo experiments) carrying the most common mutation was recorded. This promising observation paves the way for further studies to improve the benefit of the light-to-dark thermal conversion for chaperoning activity.

Amyloid Disassembly: What Can We Learn from Chaperones?

Protein aggregation and subsequent accumulation of insoluble amyloid fibrils with cross-ß structure is an intrinsic characteristic of amyloid diseases, i.e., amyloidoses. Amyloid formation involves a series of on-pathway and off-pathway protein aggregation events, leading to mature insoluble fibrils that eventually accumulate in multiple tissues. In this cascade of events, soluble oligomeric species are formed, which are among the most cytotoxic molecular entities along the amyloid cascade. The direct or indirect action of these amyloid soluble oligomers and amyloid protofibrils and fibrils in several tissues and organs lead to cell death in some cases and organ disfunction in general. There are dozens of different proteins and peptides causing multiple amyloid pathologies, chief among them Alzheimer's, Parkinson's, Huntington's, and several other neurodegenerative diseases. Amyloid fibril disassembly is among the disease-modifying therapeutic strategies being pursued to overcome amyloid pathologies. The clearance of preformed amyloids and consequently the arresting of the progression of organ deterioration may increase patient survival and quality of life. In this review, we compiled from the literature many examples of chemical and biochemical agents able to disaggregate preformed amyloids, which have been classified as molecular chaperones, chemical chaperones, and pharmacological chaperones. We focused on their mode of action, chemical structure, interactions with the fibrillar structures, morphology and toxicity of the disaggregation products, and the potential use of disaggregation agents as a treatment option in amyloidosis.

The use of pharmacological chaperones in rare diseases caused by reduced protein stability.

Rare diseases are most often caused by inherited genetic disorders that, after translation, will result in a protein with altered function. Decreased protein stability is the most frequent mechanism associated with a congenital pathogenic missense mutation and it implies the destabilization of the folded conformation in favour of unfolded or misfolded states. In the cellular context and when experimental data is available, a mutant protein with altered thermodynamic stability often also results in impaired homeostasis, with the deleterious accumulation of protein aggregates, metabolites and/or metabolic by-products. In the last decades, a significant effort has enabled the characterization of rare diseases associated to protein stability defects and triggered the development of innovative therapeutic intervention lines, say, the use of pharmacological chaperones to correct the intracellular impaired homeostasis. Here, we review the current knowledge on rare diseases caused by reduced protein stability, paying special attention to the thermodynamic aspects of the protein destabilization, also focusing on some examples where pharmacological chaperones are being tested.

Targeting trafficking as a therapeutic avenue for misfolded GPCRs leading to endocrine diseases.

G protein-coupled receptors (GPCRs) are plasma membrane proteins associated with an array of functions. Mutations in these receptors lead to a number of genetic diseases, including diseases involving the endocrine system. A particular subset of loss-of-function mutant GPCRs are misfolded receptors unable to traffic to their site of function (i.e. the cell surface plasma membrane). Endocrine disorders in humans caused by GPCR misfolding include, among others, hypo- and hyper-gonadotropic hypogonadism, morbid obesity, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, X-linked nephrogenic diabetes insipidus, congenital hypothyroidism, and familial glucocorticoid resistance. Several in vitro and in vivo experimental approaches have been employed to restore function of some misfolded GPCRs linked to endocrine disfunction. The most promising approach is by employing pharmacological chaperones or pharmacoperones, which assist abnormally and incompletely folded proteins to refold correctly and adopt a more stable configuration to pass the scrutiny of the cell's quality control system, thereby correcting misrouting. This review covers the most important aspects that regulate folding and traffic of newly synthesized proteins, as well as the experimental approaches targeted to overcome protein misfolding, with special focus on GPCRs involved in endocrine diseases.

GCase Enhancers: A Potential Therapeutic Option for Gaucher Disease and Other Neurological Disorders.

Pharmaceutical chaperones (PCs) are small compounds able to bind and stabilize misfolded proteins, allowing them to recover their native folding and thus their biological activity. In particular, lysosomal storage disorders (LSDs), a class of metabolic disorders due to genetic mutations that result in misfolded lysosomal enzymes, can strongly benefit from the use of PCs able to facilitate their translocation to the lysosomes. This results in a recovery of their catalytic activity. No PC for the GCase enzyme (lysosomal acid-ß-glucosidase, or glucocerebrosidase) has reached the market yet, despite the importance of this enzyme not only for Gaucher disease, the most common LSD, but also for neurological disorders, such as Parkinson's disease. This review aims to describe the efforts made by the scientific community in the last 7 years (since 2015) in order to identify new PCs for the GCase enzyme, which have been mainly identified among glycomimetic-based compounds.

Synthesis of a New ß-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.

GM1 gangliosidosis is a rare lysosomal disease caused by the deficiency of the enzyme ß-galactosidase (ß-Gal; GLB1; E.C. 3.2.1.23), responsible for the hydrolysis of terminal ß-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans, such as keratan-sulfate. With the aim of identifying new pharmacological chaperones for GM1 gangliosidosis, the synthesis of five new trihydroxypiperidine iminosugars is reported in this work. The target compounds feature a pentyl alkyl chain in different positions of the piperidine ring and different absolute configurations of the alkyl chain at C-2 and the hydroxy group at C-3. The organometallic addition of a Grignard reagent onto a carbohydrate-derived nitrone in the presence or absence of a suitable Lewis Acid was exploited, providing structural diversity at C-2, followed by the ring-closure reductive amination step. An oxidation-reduction process allowed access to a different configuration at C-3. The N-pentyl trihydroxypiperidine iminosugar was also synthesized for the purpose of comparison. The biological evaluation of the newly synthesized compounds was performed on leucocyte extracts from healthy donors and identified two suitable ß-Gal inhibitors, namely compounds 10 and 12. Among these, compound 12 showed chaperoning properties since it enhanced ß-Gal activity by 40% when tested on GM1 patients bearing the p.Ile51Asn/p.Arg201His mutations.

Pharmacological Chaperones for GCase that Switch Conformation with pH Enhance Enzyme Levels in Gaucher Animal Models.

Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engineering their inherent basicity, we have synthesized PCs that change conformation between the ER and the lysosomal environment, thus weakening binding to GCase after its successful trafficking to the lysosome. NMR studies confirmed the conformational change while X-ray data reveal bound conformations and binding modes. These results were further corroborated by cell studies showing increases in GCase activity when using the pH-switchable probe at low dosing. Preliminary in vivo assays with humanized mouse models of Gaucher showed enhanced GCase activity levels in relevant tissues, including the brain, further supporting their potential.

Design and synthesis of biologically active carbaglycosylamines: From glycosidase inhibitors to pharmacological chaperones.

For over 50 years, our group has been involved in synthetic studies on biologically active cyclitols including carbasugars. Among a variety of compounds synthesized, this review focuses on carbaglycosylamine glycosidase inhibitors, highlighting the following: (1) the naturally occurring N-linked carbaoligosaccharide α-amylase inhibitor acarbose and related compounds; (2) the novel synthetic ß-glycosidase inhibitors, 1'-epi-acarviosin and its 6-hydroxy analogue as well as ß-valienaminylceramide and its 4'-epimer; (3) the discovery of the ß-glycosidase inhibitors with chaperone activity, N-octyl-ß-valienamine (NOV) and its 4-epimer (NOEV); and (4) the recent development of the potential pharmacological chaperone N-alkyl-conduramine F-4 derivatives.

Alchemical Design of Pharmacological Chaperones with Higher Affinity for Phenylalanine Hydroxylase.

Phenylketonuria (PKU) is a rare metabolic disease caused by variations in a human gene, PAH, encoding phenylalanine hydroxylase (PAH), and the enzyme converting the essential amino acid phenylalanine into tyrosine. Many PKU-causing variations compromise the conformational stability of the encoded enzyme, decreasing or abolishing its catalytic activity, and leading to an elevated concentration of phenylalanine in the blood, which is neurotoxic. Several therapeutic approaches have been developed to treat the more severe manifestations of the disorder, but they are either not entirely effective or difficult to adhere to throughout life. In a search for novel pharmacological chaperones to treat PKU, a lead compound was discovered (compound IV) that exhibited promising in vitro and in vivo chaperoning activity on PAH. The structure of the PAH-IV complex has been reported. Here, using alchemical free energy calculations (AFEC) on the structure of the PAH-IV complex, we design a new generation of compound IV-analogues with a higher affinity for the enzyme. Seventeen novel analogues were synthesized, and thermal shift and isothermal titration calorimetry (ITC) assays were performed to experimentally evaluate their stabilizing effect and their affinity for the enzyme. Most of the new derivatives bind to PAH tighter than lead compound IV and induce a greater thermostabilization of the enzyme upon binding. Importantly, the correspondence between the calculated alchemical binding free energies and the experimentally determined ΔΔGb values is excellent, which supports the use of AFEC to design pharmacological chaperones to treat PKU using the X-ray structure of their complexes with the target PAH enzyme.