Investigation of the enzymatic activity of the Na,K-ATPase via isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) is shown here to be a sensitive and accurate method for assaying the steady-state enzyme activity of the Na⁺,K⁺-ATPase. Single ATP injection experiments yield an apparent enthalpy change for the ATP hydrolysis reaction catalyzed by the enzyme of −51 (± 1) kJ mol⁻¹. This value is independent of the amount of ADP accumulated in the sample cell, which indicates that under the experimental conditions studied here (saturating Na⁺ and K⁺ concentrations) ADP does not inhibit enzyme activity by reversal of the phosphorylation reaction and resynthesizing ATP. Multiple ATP injection titration experiments in which varying concentrations of ADP were initially included in the sample cell could be adequately explained by a Michaelis-Menten kinetic model incorporating noncompetitive inhibition. This suggests that ADP inhibits the enzyme by binding to more than one enzyme intermediate and inhibiting forward reactions of the enzyme. Values of K_m and K_I obtained for the fits agree with literature values obtained by other methods. Because ITC is a direct method of continually monitoring enzyme activity, it is a valuable supplement to less direct or noncontinuous methods such as colorimetric, enzyme-coupled or radioactivity-based assays.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

Purification and characterization of methylglyoxal reductase from Hansenula mrakii

Methylglyoxal reductase was purified from Hansenula mrakii IFO 0895 to a homogenous state on polyacrylamide gel electrophoresis. The enzyme consisted of a single polypeptide chain with a molecular weight of 34,000. The enzyme was specific to methylglyoxal (K_m = 1.92 mM) and NADPH (K_m = 40.8 μM). The activity of the enzyme was inhibited by p-chloromercuribenzoate and HgCl₂. NADP also inhibited the activity of the enzyme, and the K_i value was calculated to be 0.25 mM.     

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C₄ species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C₄ enzyme has a group with a pK of about 6.0 (6.6 for the C₄ enzyme), as indicated from plots of the log V_max/K_m (V_max = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C₄ enzyme-NADP-Mg²⁺ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K_m increases, but V_max remains constant. The log V_max/K_m plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K_m and a decrease in V_max for the C₄ enzyme. As in the case of the animal enzyme, the log V_max/K_m plot for the C₄ enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C₄ NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.     

α-d-Galactosidase immobilized on a soluble polymer

α-d-Galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) from green coffee beans has been immobilized by attachment to cyanogen bromide-activated Dextran T-70. Since this represents the first reported example of the preparation of a water-soluble derivative of an enzyme showing substrate inhibition, the kinetic properties, thermal stability and pH optima were investigated and compared with those of the free enzyme. The K_m, K_s, K_i, V_max, optimum substrate concentration and optimum pH were all lower than those of free enzyme. The enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation. These data, although obtained with the synthetic substrate 4-nitrophenyl-α-d-galactoside, suggest some advantages in using the enzyme conjugate for the removal of terminal α-d-galactopyranosyl groups from the erythrocyte cell surface.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme–inhibitor complex (no “tight binding”). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (k_inact). However, the inhibition constant (K_i), and therefore also the second-order inactivation rate constant k_inact/K_i, is underestimated by the traditional method by up to an order of magnitude.     

Modification of the (Na+ + K+)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate: Specific changes in enzymatic properties

Acetic anhydride irreversibly inactivated (Na⁺ + K⁺)-dependent ATPase preparations from brain, kidney, and eel electroplax. The extent of inactivation was dose dependent, and varied also with the pH of the medium, inactivation decreasing with pH in the range 8.4 to 6.7. Including KCl (k_0.5 ca. 0.6 mm) or ATP (K_0.5 ca. 1 μm) in the medium protected against inactivation, whereas MgCl₂ (k_0.5 ca. 1 mm) increased inactivation. K⁺-Dependent phosphatase activity of the enzyme was lost in parallel with (Na + K)-ATPase activity, but Na⁺-dependent phosphorylation of the enzyme and Na⁺-dependent ATPase activity were relatively resistant to inactivation. Extraction of the membrane lipids of treated enzyme preparations and replacement with exogenous lipid dispersions did not reverse the inactivation; on the other hand, the catalytic peptide of the enzyme was labeled after incubation with radioactive acetic anhydride. For the enzymatic activity remaining after treatment with acetic anhydride several kinetic properties were also modified. For the K-phosphatase reaction the k_0.5 for K⁺-activation was greatly increased, whereas for the (Na + K)-ATPase reaction the k_0.5 for neither K⁺ nor Na⁺ was increased, although the apparent k_m for ATP was decreased. These observations are interpreted in terms of a decreased apparent affinity for K⁺ at the moderate-affinity α sites of the enzyme, sites involved in (i) activating the K-phosphatase but not the (Na + K)-ATPase reactions and (ii) influencing the k_m for ATP. Effects of trinitrobenzene sulfonate (TNBS) on the enzyme preparations were similar: Both KCl and ATP reduced the extent of irreversible inactivation; the pH dependence indicated a pK_a for the reactive enzyme groups of 7.5–8; and TNBS affected K⁺-activation analogously. Moreover, inactivation by acetic anhydride and TNBS followed the pattern of mutually exclusive inhibitors, and prior treatment with TNBS reduced labeling of the enzyme by radioactive acetic anhydride. By contrast, partial inactivation by pyridoxal phosphate or N-ethylmaleimide did not result in a similarly modified enzyme. The effects of acetic anhydride and TNBS appear to be mediated (at least in part) through amino groups not accessible to or reactive with the other reagents: groups which influence the moderate-affinity α sites and which are protected by the presence of K⁺ at these sites.     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Choline sulfokinase of Penicillium chrysogenum: partial purification and kinetic mechanism.

Choline sulfokinase (3′-phosphoadenosine 5′-phosphosulfate (PAPS):choline sulfotransferase, EC 2.8.2.6) was purified approximately 30-fold from the mycelium of Penicillium chrysogenum. The K_m for PAPS is 12 μm. The enzyme is remarkably specific for the adenosine 3′,5′ (or 2′-5′)-diphosphate moiety. 3′,5′-ADP (PAP) has a K_i of 2.5 to 14 μm (depending on the choline concentration) whereas the K_i values of 3′-AMP, 5′-AMP, and 5′-ADP are at least 300-fold higher. The enzyme is also highly specific for choline (K_m = 17 μM). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate (K_m = 800 μmV = 35% of V with choline). Triethylaminoethanol was a significantly poorer substrate (K_m = 2800 μM; V = 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the maximum activity decreases and the K_m for choline increases. (The K_m for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K_m for choline) by a 10-min preincubation with 50 mm mercaptoethanol. However, prolonged incubation (24 h) with 50 mm mercaptoethanol results in irreversible denaturation. Initial velocity studies established that the enzyme follows a sequential kinetic mechanism. Product inhibition studies suggest a rapid equilibrium random binding sequence. Choline-O-phosphate (a dead-end inhibitor) is linearly competitive with choline and a linear mixed type inhibitor with respect to PAPS. Choline analogs lacking the alcohol (or ester) group (e.g., trimethylammonium, neurine, chlorocholine) are competitive dead-end inhibitors with respect to choline but are uncompetitive with respect to PAPS. Thiocholine is a linear mixed type inhibitor with respect to PAPS, but the reciprocal plots are almost parallel. These results suggest that the analogs lacking an oxygen atom have a negligible affinity for the free enzyme and bind predominantly to the enzyme-PAPS complex.     

Deep learning allows genome-scale prediction of Michaelis constants from structural features

The Michaelis constant K_M describes the affinity of an enzyme for a specific substrate and is a central parameter in studies of enzyme kinetics and cellular physiology. As measurements of K_M are often difficult and time-consuming, experimental estimates exist for only a minority of enzyme–substrate combinations even in model organisms. Here, we build and train an organism-independent model that successfully predicts K_M values for natural enzyme–substrate combinations using machine and deep learning methods. Predictions are based on a task-specific molecular fingerprint of the substrate, generated using a graph neural network, and on a deep numerical representation of the enzyme’s amino acid sequence. We provide genome-scale K_M predictions for 47 model organisms, which can be used to approximately relate metabolite concentrations to cellular physiology and to aid in the parameterization of kinetic models of cellular metabolism.

To understand the action of an enzyme, we need to know its affinity for its substrates, quantified by Michaelis constants, but these are difficult to measure experimentally. This study shows that a deep learning model that can predict them from structural features of the enzyme and substrate, providing KM predictions for all enzymes across 47 model organisms.     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

The kinetics of rat liver citrate synthase in situ.

The kinetics of citrate synthase in situ in toluene-treated rat liver mitochondria were studied. The V_max, K_m, and kinetic pattern for oxaloacetate were the same as those for the pure rat liver citrate synthase. The K_m, for acetyl-CoA for the in situ enzyme was increased compared to pure enzyme (8.5 to 77 μm), and the sensitivity of the in situ enzyme to inhibition by ATP, NADPH, or tricarballylate was decreased. The change seen with ATP was not due to problems of small molecule diffusion.     

Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings

Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a K_m for acetyl-CoA of 74 micromolar and a V_max of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a K_is of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate.     

Antibody binding of cartilage-specific proteoglycans

The spectroscopically observable acid dissociation constant of aspartate aminotransferase (EC 2.6.1.1) varies to different degrees upon the addition of different monovalent anions. These interactions may be described by the minimal scheme

where XEH and XE represent anion complexes with the acidic (EH) and basic (E) forms of the enzyme, respectively. Both graphical and computer procedures were used to determine the three equilibrium constants which describe such a system. The analysis was based upon the effects of salt concentration (X) upon the apparent pK′_a of the enzyme determined spectrophotometrically. The affinities for anions of the basic enyzme are less than those of the acidic form of the enzyme so that the apparent pK′_a rises with anion concentration to a limiting value; pK₄ of the enzyme anion complex. That different anion-enzyme complexes have different pK₄'s reflects the fact that the interaction specificities of the basic and acidic enzymes differ. Cacodylate did not appear either to cause significant effects on the chromophoric pK's or to compete with the binding of halide or carboxylate anions which cause a perturbation of the pK_a.     

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant Plasmodium chabaudi

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant smodium chabaudi. International Journal for Parasitology16: 483–490. Thymidylate synthetase from cloned strains of pyrimethamine-sensitive and resistant P. chubaudi were partially purified and characterized. The enzyme from both strains have equal mol. wt of 120,000 as estimated by Sephadex G-200 column chromatography. The enzyme from drug-sensitive parasites has an optimum pH of 6.5–7.5 and is stable at pH 4–11 while that from drug-resistant strain has an pH optimum of 7.0–8.0 and is stable at pH 5–10. The K_m for methylenetetrahydrofolate are 206 ± 6 and 495 ± 5 μm for the enzyme from drug-resistant and sensitive parasites, respectively. The K_m for dUMP of the enzyme from drug-resistant and sensitive parasites are 42 ± 1 and 49 ± 6 μm, respectively. Inhibition of the enzyme from both strains by FdUMP are competitive with dUMP; however,the K_is for the enzyme from drug-resistant strain (0.043 ± 0.005 μm) is less than that from drug-sensitive strain (0.11 ± 0.007 μm) by a factor of 2.5. The K_ii for methotrexate with respect to methylenetetrahydrofolate of the enzyme from drug-resistant parasites (58 ± 3 μm) is 3 times larger than that from drug-sensitive parasites (17 ± 1 μm).     

Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K_m and V_max values of 0.25 mM and 5.35 μkat.mg^?1 enzyme protein. Thus, the enzyme had a very high (292,000 M^?1.s^?1) catalytic efficiency (K_cat/K_m). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol^?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K_i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.     

Properties of Phosphoglycolate Phosphatase from Chlamydomonas reinhardtii and Anacystis nidulans

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO₂ in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K_m of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K_m of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.

The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co²⁺, but was also activated by Mg²⁺ (K_a = 30 micromolar), Mn²⁺, and Zn²⁺. The Anacystis enzyme was most effectively activated by Mg²⁺ (K_a = 140 micromolar), and was also activated by Co²⁺ and Mn²⁺, but not by Zn²⁺. Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K_a = 140 micromolar), bromide, nitrate, bicarbonate (K_a = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K_a = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.

The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.