Protein Tyrosine Phosphatase regulation by Reactive Oxygen Species.

Protein Tyrosine Phosphatases (PTPs) help to maintain the balance of protein phosphorylation signals that drive cell division, proliferation, and differentiation. These enzymes are also well-suited to redox-dependent signaling and oxidative stress response due to their cysteine-based catalytic mechanism, which requires a deprotonated thiol group at the active site. This review focuses on PTP structural characteristics, active site chemical properties, and vulnerability to change by reactive oxygen species (ROS). PTPs can be oxidized and inactivated by H2O2 through three non-exclusive mechanisms. These pathways are dependent on the coordinated actions of other H2O2-sensitive proteins, such as peroxidases like Peroxiredoxins (Prx) and Thioredoxins (Trx). PTPs undergo reversible oxidation by converting their active site cysteine from thiol to sulfenic acid. This sulfenic acid can then react with adjacent cysteines to form disulfide bonds or with nearby amides to form sulfenyl-amide linkages. Further oxidation of the sulfenic acid form to the sulfonic or sulfinic acid forms causes irreversible deactivation. Understanding the structural changes involved in both reversible and irreversible PTP oxidation can help with their chemical manipulation for therapeutic intervention. Nonetheless, more information remains unidentified than is presently known about the precise dynamics of proteins participating in oxidation events, as well as the specific oxidation states that can be targeted for PTPs. This review summarizes current information on PTP-specific oxidation patterns and explains how ROS-mediated signal transmission interacts with phosphorylation-based signaling machinery controlled by growth factor receptors and PTPs.

Biochemical characterization of the Eya and PP2A-B55α interaction.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

Enzyme mechanistic studies of NMA1982, a protein tyrosine phosphatase and potential virulence factor in Neisseria meningitidis.

Protein phosphorylation is an integral part of many cellular processes, not only in eukaryotes but also in bacteria. The discovery of both prokaryotic protein kinases and phosphatases has created interest in generating antibacterial therapeutics that target these enzymes. NMA1982 is a putative phosphatase from Neisseria meningitidis, the causative agent of meningitis and meningococcal septicemia. The overall fold of NMA1982 closely resembles that of protein tyrosine phosphatases (PTPs). However, the hallmark C(X)5R PTP signature motif, containing the catalytic cysteine and invariant arginine, is shorter by one amino acid in NMA1982. This has cast doubt about the catalytic mechanism of NMA1982 and its assignment to the PTP superfamily. Here, we demonstrate that NMA1982 indeed employs a catalytic mechanism that is specific to PTPs. Mutagenesis experiments, transition state inhibition, pH-dependence activity, and oxidative inactivation experiments all support that NMA1982 is a genuine PTP. Importantly, we show that NMA1982 is secreted by N. meningitidis, suggesting that this protein is a potential virulence factor. Future studies will need to address whether NMA1982 is indeed essential for N. meningitidis survival and virulence. Based on its unique active site conformation, NMA1982 may become a suitable target for developing selective antibacterial drugs.

Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a ß site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the ß site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

The progress of research into pseudophosphatases.

Pseudophosphatases are a class of phosphatases that mutate at the catalytically active site. They play important parts in many life processes and disorders, e.g., cell apoptosis, stress reaction, tumorigenesis, axon differentiation, Charcot-Marie-Tooth, and metabolic dysfunction. The present review considers the structures and action types of pseudophosphatases in four families, protein tyrosine phosphatases (PTPs), myotube protein phosphatases (MTMs), phosphatases and tensin homologues (PTENs) and dual specificity phosphatases (DUSPs), as well as their mechanisms in signaling and disease. We aimed to provide reference material for the research and treatment of related diseases.

Crystal structure of the catalytic domain of human RPTPH.

Receptor-type protein tyrosine phosphatases (RPTPs) receive extracellular stimuli and transfer them into cells. They regulate cell growth, differentiation and death via specific signals. They have also been implicated in cancer, diabetes and neurological diseases. RPTPH, a member of the type 3 RPTP (R3-PTP) family, is an important regulator of colorectal cancer and hepatic carcinoma. Despite its importance in drug development, the structure of RPTPH has not yet been resolved. Here, the crystal structure of the catalytic domain of RPTPH was determined at 1.56 Šresolution. Despite similarities to other R3-PTPs in its overall structure, RPTPH exhibited differences in its loop regions and side-chain conformations. Compared with other R3-PTPs, RPTPH has unique side chains near its active site that may confer specificity for inhibitor binding. Therefore, detailed information on the structure of RPTPH provides clues for the development of specific inhibitors.

Crystal structure of the Cys-NO modified YopH tyrosine phosphatase.

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.

Structure-activity relationship studies of allosteric inhibitors of EYA2 tyrosine phosphatase.

Human eyes absent (EYA) proteins possess Tyr phosphatase activity, which is critical for numerous cancer and metastasis promoting activities, making it an attractive target for cancer therapy. In this work, we demonstrate that the inhibitor-bound form of EYA2 does not favour binding to Mg2+ , which is indispensable for the Tyr phosphatase activity. We further describe characterization and optimization of this class of allosteric inhibitors. A series of analogues were synthesized to improve potency of the inhibitors and to elucidate structure-activity relationships. Two co-crystal structures confirm the binding modes of this class of inhibitors. Our medicinal chemical, structural, biochemical, and biophysical studies provide insight into the molecular interactions of EYA2 with these allosteric inhibitors. The compounds derived from this study are useful for exploring the function of the Tyr phosphatase activity of EYA2 in normal and cancerous cells and serve as reference compounds for screening or developing allosteric phosphatase inhibitors. Finally, the co-crystal structures reported in this study will aid in structure-based drug discovery against EYA2.

Recent advances in the development of allosteric protein tyrosine phosphatase inhibitors for drug discovery.

Protein tyrosine phosphatases (PTPs) superfamily catalyzes tyrosine de-phosphorylation which affects a myriad of cellular processes. Imbalance in signal pathways mediated by PTPs has been associated with development of many human diseases including cancer, metabolic, and immunological diseases. Several compelling evidence suggest that many members of PTP family are novel therapeutic targets. However, the clinical development of conventional PTP-based active-site inhibitors originally was hampered by the poor selectivity and pharmacokinetic properties. In this regard, PTPs has been widely dismissed as "undruggable." Nonetheless, allosteric modulation has become increasingly an influential and alternative approach that can be exploited for drug development against PTPs. Unlike active-site inhibitors, allosteric inhibitors exhibit a remarkable target-selectivity, drug-likeness, potency, and in vivo activity. Intriguingly, there has been a high interest in novel allosteric PTPs inhibitors within the last years. In this review, we focus on the recent advances of allosteric inhibitors that have been explored in drug discovery and have shown an excellent result in the development of PTPs-based therapeutics. A special emphasis is placed on the structure-activity relationship and molecular mechanistic studies illustrating applications in chemical biology and medicinal chemistry.

Reassessment of the Proteomic Composition and Function of Extracellular Vesicles in the Seminal Plasma.

Seminal plasma contains a high concentration of extracellular vesicles (EVs). The heterogeneity of small EVs or the presence of nonvesicular extracellular matter (NV) pose major obstacles in understanding the composition and function of seminal EVs. In this study, we employed high-resolution density gradient fractionation to accurately characterize the composition and function of seminal EVs and NV. We found that the seminal EVs could be divided into 3 different subtypes-namely, high-density EV (EV-H), medium-density EV (EV-M), and low-density EV (EV-L)-after purification using iodixanol, while NV was successfully isolated. EVs and NV display different features in size, shape, and expression of some classic exosome markers. Both EV-H and NV could markedly promote sperm motility and capacitation compared with EV-M and EV-L, whereas only the NV fraction induced sperm acrosome reaction. Proteomic analysis results showed that EV-H, EV-M, EV-L, and NV had different protein components and were involved in different physiological functions. Further study showed that EV-M might reduce the production of sperm intrinsic reactive oxygen species through glutathione S-transferase mu 2. This study provides novel insights into important aspects of seminal EVs constituents and sounder footing to explore their functional properties in male fertility.

A New Paradigm for KIM-PTP Drug Discovery: Identification of Allosteric Sites with Potential for Selective Inhibition Using Virtual Screening and LEI Analysis.

The kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), HePTP, PTPSL and STEP, are involved in the negative regulation of mitogen-activated protein kinase (MAPK) signalling pathways and are important therapeutic targets for a number of diseases. We have used VSpipe, a virtual screening pipeline, to identify a ligand cluster distribution that is unique to this subfamily of PTPs. Several clusters map onto KIM-PTP specific sequence motifs in contrast to the cluster distribution obtained for PTP1B, a classic PTP that mapped to general PTP motifs. Importantly, the ligand clusters coincide with previously reported functional and substrate binding sites in KIM-PTPs. Assessment of the KIM-PTP specific clusters, using ligand efficiency index (LEI) plots generated by the VSpipe, ascertained that the binders in these clusters reside in a more drug-like chemical-biological space than those at the active site. LEI analysis showed differences between clusters across all KIM-PTPs, highlighting a distinct and specific profile for each phosphatase. The most druggable cluster sites are unexplored allosteric functional sites unique to each target. Exploiting these sites may facilitate the delivery of inhibitors with improved drug-like properties, with selectivity amongst the KIM-PTPs and over other classical PTPs.

Genome-wide survey of tyrosine phosphatases in thirty mammalian genomes.

The age of genomics has given us a wealth of information and the tools to study whole genomes. This, in turn, has facilitated genome-wide studies among organisms that were relatively less studied in the pre-genomic era or are non-model organisms. This paves the way to the discovery of interesting evolutionary patterns, which are brought to light by genome-wide surveys of protein superfamilies. Phosphorylation is a post-translational modification that is utilised across all clades of life, and acts as an important signalling switch, regulating several cellular processes. Tyrosine phosphatases, which are found predominantly in eukaryotes, act on phosphorylated tyrosine residues and sometimes on other substrates. Extending on our previous effort to look for tyrosine phosphatases in the human genome, we have looked for sequences of the cysteine-based tyrosine phosphatase superfamily in thirty mammalian genomes from all across Mammalia and validated the sequences with the presence of the signature catalytic motif. Domain architecture annotation, followed by in-depth analysis, revealed interesting taxon-specific patterns such as subtle differences between the protein families in marsupials and early mammals versus placental mammals. Finally, we discuss an interesting case of loss of the tyrosine phosphatase domain from a gene product in the course of eutherian evolution.

Characterization of low molecular weight protein tyrosine phosphatases of Entamoeba histolytica.

Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).

Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases.

Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) perform dephosphorylation to regulate signaling pathways in cellular responses. The hydrogen bonding network in their active site plays an important conformational role and supports the phosphatase activity. Nearly half of dual-specificity phosphatases (DUSPs) use three conserved residues, including aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop, to form the hydrogen bonding network, the D-, P-, N-triloop interaction (DPN-triloop interaction). In this study, DUSP22 is used to investigate the importance of the DPN-triloop interaction in active site formation. Alanine mutations and somatic mutations of the conserved residues, D57, S93, and N128 substantially decrease catalytic efficiency (kcat/KM) by more than 102-fold. Structural studies by NMR and crystallography reveal that each residue can perturb the three loops and induce conformational changes, indicating that the hydrogen bonding network aligns the residues in the correct positions for substrate interaction and catalysis. Studying the DPN-triloop interaction reveals the mechanism maintaining phosphatase activity in N-loop-containing PTPs and provides a foundation for further investigation of active site formation in different members of this protein class.

A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2.

The cyclin M (CNNM) family of Mg2+ transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable Kd. We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inhibited the changes in FRET intensity, whereas non-YPet-tagged CNNM3 with a mutation at the PRL2-binding site did not exhibit such inhibition. Furthermore, newly synthesized peptides derived from the CNNM loop region, with the PRL-binding sequences of the CNNM3 CBS domain, inhibited the interactions between CNNM3 and PRL2. Overall, these results showed that this method can be used for screening to identify inhibitors of CNNM-PRL interactions, potentially for novel anticancer therapy.

In Vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine Phosphatases in Cells.

The reversible oxidation of protein tyrosine phosphatases (PTPs) impairs their ability to dephosphorylate substrates in vivo. This transient inactivation of PTPs occurs as their conserved catalytic cysteine residue reacts with cellular oxidants thereby abolishing the ability of this reactive cysteine to attack the phosphate of the target substrate. Hence, in vivo, the inhibition of specific PTPs in response to regulated and localized rises in cellular oxidants enables phospho-dependent signaling. We present assays that measure the endogenous activity of specific PTPs that become transiently inactivated in cells exposed to growth factors. Here, we describe the methods and highlight the pitfalls to avoid post-lysis oxidation of PTPs in order to assess the inactivation and the reactivation of PTPs targeted by cellular oxidants in signal transduction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Cell transfection (optional) Support Protocol: Preparation of degassed lysis buffers Basic Protocol 2: Cellular extraction in anaerobic conditions Basic Protocol 3: Enrichment and activity assay of specific PTPs Alternate Protocol: Measurement of active PTPs via direct cysteinyl labeling.

PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.

Phosphatases of regenerating liver (PRLs) are markers of cancer and promote tumor growth. They have been implicated in a variety of biochemical pathways but the physiologically relevant target of phosphatase activity has eluded 20 years of investigation. Here, we show that PRL3 catalytic activity is not required in a mouse model of metastasis. PRL3 binds and inhibits CNNM4, a membrane protein associated with magnesium transport. Analysis of PRL3 mutants specifically defective in either CNNM-binding or phosphatase activity demonstrate that CNNM binding is necessary and sufficient to promote tumor metastasis. As PRLs do have phosphatase activity, they are in fact pseudo-pseudophosphatases. Phosphatase activity leads to formation of phosphocysteine, which blocks CNNM binding and may play a regulatory role. We show levels of PRL cysteine phosphorylation vary in response to culture conditions and in different tissues. Examination of related protein phosphatases shows the stability of phosphocysteine is a unique and evolutionarily conserved property of PRLs. The demonstration that PRL3 functions as a pseudophosphatase has important ramifications for the design of PRL inhibitors for cancer.

RPTPα phosphatase activity is allosterically regulated by the membrane-distal catalytic domain.

Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.

Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells.

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation-during which the PTP enzymes act on the peptide substrate-the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.

Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays.

The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This Research Article describes the use of phosphotyrosine-containing peptide arrays together with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to directly profile phosphatase substrate selectivities. Twenty-two protein tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of a R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level and which this work suggests may be due to an increased activity toward phosphatase enzymes. The use of self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.