The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives.

The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within the context of present waste management practices. This review reports on the prospective of biotechnological tools for plastic bio-recycling within the framework of plastic waste management in Europe. Available biotechnology tools can support polyethylene terephthalate (PET) recycling. However, PET represents only ≈7% of unrecycled plastic waste. Polyurethanes, the principal unrecycled waste fraction, together with other thermosets and more recalcitrant thermoplastics (e.g., polyolefins) are the next plausible target for enzyme-based depolymerization, even if this process is currently effective only on ideal polyester-based polymers. To extend the contribution of biotechnology to plastic circularity, optimization of collection and sorting systems should be considered to feed chemoenzymatic technologies for the treatment of more recalcitrant and mixed polymers. In addition, new bio-based technologies with a lower environmental impact in comparison with the present approaches should be developed to depolymerize (available or new) plastic materials, that should be designed for the required durability and for being susceptible to the action of enzymes.

The Protein Imager: a full-featured online molecular viewer interface with server-side HQ-rendering capabilities.

SUMMARY: Molecular viewers' long learning curve is hindering researchers in approaching the field of structural biology for the first time. Herein, we present 'The Protein Imager', a lightweight, powerful and easy-to-use interface as a next-gen online molecular viewer. Furthermore, the interface is linked to an automated server-side rendering system able to generate publication-quality molecular illustrations. The Protein Imager interface has been designed for easy usage for beginners and experts in the field alike. The interface allows the preparation of very complex molecular views maintaining a high level of responsiveness even on mobile devices. AVAILABILITY AND IMPLEMENTATION: The Protein Imager interface is freely available online at https://3dproteinimaging.com/protein-imager. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL-1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL-1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 µg of enzyme mL-1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

Advances in Enzymatic Synthesis of D-Amino Acids.

In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these "unnatural" molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes.

Succinic Semialdehyde Dehydrogenase Deficiency: In Vitro and In Silico Characterization of a Novel Pathogenic Missense Variant and Analysis of the Mutational Spectrum of ALDH5A1.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes.

l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic l-amino acids into α-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel α+ß subdomain placed close to the putative transmembrane α-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

Structure-function relationships in human d-amino acid oxidase variants corresponding to known SNPs.

In the brain, d-amino acid oxidase plays a key role in modulating the N-methyl-d-aspartate receptor (NMDAR) activation state, catalyzing the stereospecific degradation of the coagonist d-serine. A relationship between d-serine signaling deregulation, NMDAR dysfunction, and CNS diseases is presumed. Notably, the R199W substitution in human DAAO (hDAAO) was associated with familial amyotrophic lateral sclerosis (ALS), and further coding substitutions, i.e., R199Q and W209R, were also deposited in the single nucleotide polymorphism database. Here, we investigated the biochemical properties of these different hDAAO variants. The W209R hDAAO variant shows an improved d-serine degradation ability (higher activity and affinity for the cofactor FAD) and produces a greater decrease in cellular d/(d+l) serine ratio than the wild-type counterpart when expressed in U87 cells. The production of H2O2 as result of excessive d-serine degradation by this hDAAO variant may represent the factor affecting cell viability after stable transfection. The R199W/Q substitution in hDAAO altered the protein conformation and enzymatic activity was lost under conditions resembling the cellular ones: this resulted in an abnormal increase in cellular d-serine levels. Altogether, these results indicate that substitutions that affect hDAAO functionality directly impact on d-serine cellular levels (at least in the model cell system used). The pathological effect of the expression of the R199W hDAAO, as observed in familial ALS, originates from both protein instability and a decrease in kinetic efficiency: the increase in synaptic d-serine may be mainly responsible for the neurotoxic effect. This information is expected to drive future targeted treatments.

Aminoacetone oxidase from Streptococcus oligofermentans belongs to a new three-domain family of bacterial flavoproteins.

The aaoSo gene from Streptococcus oligofermentans encodes a 43 kDa flavoprotein, aminoacetone oxidase (SoAAO), which was reported to possess a low catalytic activity against several different L-amino acids; accordingly, it was classified as an L-amino acid oxidase. Subsequently, SoAAO was demonstrated to oxidize aminoacetone (a pro-oxidant metabolite), with an activity ~25-fold higher than the activity displayed on L-lysine, thus lending support to the assumption of aminoacetone as the preferred substrate. In the present study, we have characterized the SoAAO structure-function relationship. SoAAO is an FAD-containing enzyme that does not possess the classical properties of the oxidase/dehydrogenase class of flavoproteins (i.e. no flavin semiquinone formation is observed during anaerobic photoreduction as well as no reaction with sulfite) and does not show a true L-amino acid oxidase activity. From a structural point of view, SoAAO belongs to a novel protein family composed of three domains: an α/ß domain corresponding to the FAD-binding domain, a ß-domain partially modulating accessibility to the coenzyme, and an additional α-domain. Analysis of the reaction products of SoAAO on aminoacetone showed 2,5-dimethylpyrazine as the main product; we propose that condensation of two aminoacetone molecules yields 3,6-dimethyl-2,5-dihydropyrazine that is subsequently oxidized to 2,5-dimethylpyrazine. The ability of SoAAO to bind two molecules of the substrate analogue O-methylglycine ligand is thought to facilitate the condensation reaction. A specialized role for SoAAO in the microbial defence mechanism related to aminoacetone catabolism through a pathway yielding dimethylpyrazine derivatives instead of methylglyoxal can be proposed.

Characterization of human DAAO variants potentially related to an increased risk of schizophrenia.

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the "structurally ambivalent" peptide V47-L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d+l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57ßS/H70ßS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a ß-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1ß), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

Cephalosporin C acylase: dream and(/or) reality.

Cephalosporins currently constitute the most widely prescribed class of antibiotics and are used to treat diseases caused by both Gram-positive and Gram-negative bacteria. Cephalosporins contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C (CephC). The 7-ACA nucleus is not sufficiently potent for clinical use; however, a series of highly effective antibiotic agents could be produced by modifying the side chains linked to the 7-ACA nucleus. The industrial production of higher-generation semi-synthetic cephalosporins starts from 7-ACA, which is obtained by deacylation of the naturally occurring antibiotic CephC. CephC can be converted to 7-ACA either chemically or enzymatically using D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase. Both these methods show limitation, including the production of toxic waste products (chemical process) and the expense (the enzymatic one). In order to circumvent these problems, attempts have been undertaken to design a single-step means of enzymatically converting CephC to 7-ACA in the course of the past 10 years. The most suitable approach is represented by engineering the activity of a known glutaryl-7-aminocephalosporanic acid acylase such that it will bind and deacylate CephC more preferentially over glutaryl-7-aminocephalosporanic acid. Here, we describe the state of the art in the production of an effective and specific CephC acylase.

L-amino acid oxidase as biocatalyst: a dream too far?

L-amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the "true" LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with "narrow" or "strict" substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.

A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC-cutinase highlights the key role of residue 243.

Enzymatic degradation of poly(ethylene terephthalate) (PET) is becoming a reality because of the identification of novel PET-hydrolysing enzymes (PHEs) and the engineering of evolved enzyme variants. Here, improved variants of leaf-branch compost cutinase (LCC), a thermostable enzyme isolated by a metagenomic approach, were generated by a semi-rational protein engineering approach. Starting from a deleted LCC form lacking the secretion signal (ΔLCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T ΔLCC variant partially (~ 67%) depolymerized amorphous PET film producing ~ 21.9 mm of products after 27 h of reaction at 72 °C. The S101N/F243T ΔLCC double variant reached a further increase in activity on PET. Notably, for both single and double variants the highest conversion yield was obtained at 55 °C. Kinetics studies and molecular dynamics simulations support that a slight decreased affinity for PET is responsible for the superior degradation performance of the S101N/F243T variant and that this stems from a slightly higher flexibility of the active site region close to position 243. Furthermore, our findings question the need for a high reaction temperature for PET degradation, at least for LCC: at ≥ 70 °C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T ΔLCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in ≤ 3 days at 55 °C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process.

On the regulation of human D-aspartate oxidase.

The human flavoenzyme D-aspartate oxidase (hDASPO) controls the level of D-aspartate in the brain, a molecule acting as an agonist of NMDA receptors and modulator of AMPA and mGlu5 receptors. hDASPO-induced D-aspartate degradation prevents age-dependent deterioration of brain functions and is related to psychiatric disorders such as schizophrenia and autism. Notwithstanding this crucial role, less is known about hDASPO regulation. Here, we report that hDASPO is nitrosylated in vitro, while no evidence of sulfhydration and phosphorylation is apparent: nitrosylation affects the activity of the human flavoenzyme to a limited extent. Furthermore, hDASPO interacts with the primate-specific protein pLG72 (a well-known negative chaperone of D-amino acid oxidase, the enzyme deputed to D-serine degradation in the human brain), yielding a ~114 kDa complex, with a micromolar dissociation constant, promoting the flavoenzyme inactivation. At the cellular level, pLG72 and hDASPO generate a cytosolic complex: the expression of pLG72 negatively affects the hDASPO level by reducing its half-life. We propose that pLG72 binding may represent a protective mechanism aimed at avoiding cytotoxicity due to H2 O2 produced by the hDASPO enzymatic degradation of D-aspartate, especially before the final targeting to peroxisomes.

Revisitation of the ßCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms.

D-amino acid oxidase (DAAO) from pig has been reported to catalyze the ß-elimination of Cl(-) from ßCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-ßCl-D-alanine and reduced enzyme-ßCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. ß-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-ßCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail.