Doxorubicin inhibits the phosphate-transport protein reconstituted in liposomes. A study on the mechanism of the inhibition

Using Thr(P)-inhibitor-1 and Ser(P)-casein as substrates, studies on the activation of calcineurin purified from bovine brain have been carried out. The phosphatase requires the synergistic action of Ca2+, calmodulin and another divalent cation (Mg2+, Mn2+, Co2+ or Ni2+, but not Zn2+) for full expression of its activity. Ca2+ and Ca2+ X calmodulin act as allosteric activators to transform the phosphatase to a relaxed conformation, while Mg2+ acts solely as a cofactor for the catalytic action of the enzyme. In addition to their function as cofactors for catalysis, transition metal ions can also substitute for Ca2+ as allosteric activators. Ca2+ and calmodulin exert their activating effects mainly by increasing the Vm of the phosphatase reaction with little effect on the Km values for the substrates or on the KA values for the divalent cation cofactors. The predominant factor in dictating the catalytic properties of calcineurin is the divalent cation cofactor. For example, with Mg2+ as a cofactor, the phosphatase exhibits an optimum around pH 8.0-8.5; while with a transition metal ion as a cofactor, the optimum is around pH 7.0-7.5, regardless of whether Thr(P)-inhibitor-1 or Ser(P)-casein serves as a substrate, in the absence or the presence of Ca2+ X calmodulin.     

Three diadenosine 5',5'-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: a specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase

Two new enzymes that hydrolyze diadenosine tetraphosphate (Ap4A) have been isolated from the acellular slime mold Physarum polycephalum. Both enzymes are different from the Physarum Ap4A symmetrical pyrophosphohydrolase previously described on the basis of their substrate specificities, reaction products, molecular weights, and divalent cation requirements. One enzyme is a nucleotide pyrophosphatase that asymmetrically hydrolyzes Ap4A to AMP and ATP. This enzyme hydrolyzes several mono- and dinucleotides with the corresponding nucleotide monophosphate as one of the products. The percentage hydrolysis of NAD+, Ap4A, and Ap4G, each at 10 microM, was 100, 56, and 51, respectively. A divalent cation is required for activity, with Ca2+ yielding 20-30 times greater activity than Mg2+ or Mn2+. Values of Km for Ap4A and Vmax are similar to the corresponding values for Ap4A symmetrical pyrophosphohydrolase. The second enzyme is a phosphodiesterase I with broad substrate reactivity. This enzyme also asymmetrically hydrolyzes Ap4A, but it does not hydrolyze NAD+. Activity of the phosphodiesterase I is stimulated by divalent cations, with Ca2+ being 50-60 times more stimulatory than Mg2+ or Mn2+. The apparent molecular weights of the nucleotide pyrophosphatase and phosphodiesterase are 184,000 and 45,000, respectively. In contrast, the Ap4A pyrophosphohydrolase hydrolyzes Ap4A to ADP, is inhibited by Ca2+ and other divalent cations, and has an apparent molecular weight of 26,000 as previously reported.     

Structural characterization of a ribonuclease III processing signal.

The structure of a ribonuclease III processing signal from bacteriophage T7 was examined by NMR spectroscopy, optical melting, and chemical and enzymatic modification. A 41 nucleotide variant of the T7 R1.1 processing signal has two Watson-Crick base-paired helices separated by an internal loop, consistent with its predicted secondary structure. The internal loop is neither rigidly structured nor completely exposed to solvent, and seems to be helical. The secondary structure of R1.1 RNA is largely insensitive to the monovalent cation concentration, which suggests that the monovalent cation sensitivity of secondary site cleavage by RNase III is not due to a low salt-induced RNA conformational change. However, spectroscopic data show that Mg2+ affects the conformation of the internal loop, suggesting a divalent cation binding site(s) within this region. The Mg(2+)-dependence of RNase III processing of some substrates may reflect not only a requirement for a divalent cation as a catalytic cofactor, but also a requirement for a local RNA conformation which is divalent cation-stabilized.     

Human hypoxanthine guanine phosphoribosyltransferase. The role of magnesium ion in a phosphoribosylpyrophosphate-utilizing enzyme

The role of Mg ions in the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction have been studied using accurate values of proton and Mg stability constants of phosphoribosylpyrophosphate (P-Rib-PP) determined from pH titration data. The results obtained favor the conclusion that the dimagnesium salt of P-Rib-PP is the true substrate of the enzyme. The other species of P-Rib-PP do not appreciably affect the initial reaction rate. The inhibition of the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction observed at high MgCl2 concentration can be attributed to a competitive inhibition of Mg2+ with respect to the dimagnesium salt of P-Rib-PP, suggesting that these ionic species bind to the same enzyme form. At a fixed [P-Rib-PPtot], the concentration of its dimagnesium complex is a sigmoidal function of MgCl2 concentration, suggesting that caution must be employed in the interpretation of sigmoidal saturation curves for P-Rib-PP-utilizing enzymes when low and not constant concentrations of the divalent cation are used.     

Determination of the metal ion dependence and substrate specificity of a hydratase involved in the degradation pathway of biphenyl/chlorobiphenyl

BphH is a divalent metal ion-dependent hydratase that catalyzes the formation of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate (HPDA). This reaction lies on the catabolic pathway of numerous aromatics, including the significant environmental pollutant, polychlorinated biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia xenoverans LB400, was overexpressed and purified to homogeneity. Atomic absorption spectroscopy and Scatchard analysis reveal that only one divalent metal ion is bound to each enzyme subunit. The enzyme exhibits the highest activity when Mg2+ was used as cofactor. Other divalent cations activate the enzyme in the following order of effectiveness: Mg2+ > Mn2+ > Co2+ > Zn2+ > Ca2+. This differs from the metal activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy of the Co2+-BphH complex indicates that the divalent metal ion is hexa-coordinated in the enzyme. The nature of the metal ion affected only the kcat and not the Km values in the BphH hydration of HPDA, suggesting that cation has a catalytic rather than just a substrate binding role. BphH is able to transform alternative substrates substituted with methyl- and chlorine groups at the 5-position of HPDA. The specificity constants (kcat/Km) for 5-methyl and 5-chloro substrates are, however, lowered by eight- and 67-fold compared with the unsubstituted substrate. Significantly, kcat for the chloro-substituted substrate is eightfold lower compared with the methyl-substituted substrate, showing that electron withdrawing substituent at the 5-position of the substrate has a negative influence on enzyme catalysis.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Divalent cation-induced changes in conformation of protein kinase C

Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.     

Stereochemical probes of the argininosuccinate synthetase reaction

The stereochemical course of the argininosuccinate synthetase reaction has been determined. The SP isomer of [alpha-17O,alpha-18O,alpha beta-18O]ATP is cleaved to (SP)-[16O,17O,18O]AMP by the action of argininosuccinate synthetase in the presence of citrulline and aspartate. The overall stereochemical transformation is therefore net inversion, and thus the enzyme does not catalyze the formation of an adenylylated enzyme intermediate prior to the synthesis of citrulline adenylate. The RP isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) is a substrate in the presence of Mg2+, but the SP isomer is a substrate when Cd2+ is used as the activating divalent cation. Therefore, the lambda screw sense configuration of the beta,gamma-bidentate metal--ATP complex is preferred by the enzyme as the actual substrate. No significant discrimination could be detected between the RP and SP isomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) when Mg2+ or Mn2+ are used as the divalent cation. Argininosuccinate synthetase has been shown to require a free divalent cation for full activity in addition to the metal ion needed to complex the ATP used in the reaction.     

Interaction of Tl+ with product complexes of fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase requires divalent cations (Mg2+, Mn2+, or Zn2+) for catalysis, but a diverse set of monovalent cations (K+, Tl+, Rb+, or NH(4)(+)) will further enhance enzyme activity. Here, the interaction of Tl+ with fructose-1,6-bisphosphatase is explored under conditions that support catalysis. On the basis of initial velocity kinetics, Tl+ enhances catalysis by 20% with a K(a) of 1.3 mm and a Hill coefficient near unity. Crystal structures of enzyme complexes with Mg2+, Tl+, and reaction products, in which the concentration of Tl+ is 1 mm or less, reveal Mg2+ at metal sites 1, 2, and 3 of the active site, but little or no bound Tl+. Intermediate concentrations of Tl+ (5-20 mm) displace Mg2+ from site 3 and the 1-OH group of fructose 6-phosphate from in-line geometry with respect to bound orthophosphate. Loop 52-72 appears in a new conformational state, differing from its engaged conformation by disorder in residues 61-69. Tl+ does not bind to metal sites 1 or 2 in the presence of Mg2+, but does bind to four other sites with partial occupancy. Two of four Tl+ sites probably represent alternative binding sites for the site 3 catalytic Mg2+, whereas the other sites could play roles in monovalent cation activation.     

Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase

Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co2+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the V for oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the pKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mg2+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity.     

Subunit interaction elicited by partial inactivation with L-methionine sulfoximine and ATP differently affects the biosynthetic and gamma-glutamyltransferase reactions catalyzed by yeast glutamine synthetase

Yeast glutamine synthetase can be irreversibly inactivated in the presence of L-methionine sulfoximine, ATP, and a divalent cation Mn2+ or Mg2+. Kinetic studies with partially inactivated enzymes show that inactivation of a given subunit in the octameric glutamine synthetase affects the activities of its neighboring subunit such that the rate of the inactivation as well as the gamma-glutamyltransferase activity of the noninactivated subunits decreases while their biosynthetic activity is enhanced. This outcome of subunit interaction is the same irrespective of whether Mn2+ or Mg2+ is used to fulfill the divalent cation requirement of glutamine synthetase for the inactivation reaction and the gamma-glutamyltransferase reaction. Although only Vmax is affected in the gamma-glutamyltransferase assay, both Km (glutamate) and Vmax are changed in the biosynthetic assay.     

Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer

Glucose oxidase (GOD) from Aspergillus niger is a dimeric enzyme having high localization of negative charges on the enzyme surface and at the dimer interface. The monovalent cations induce compaction of the native conformation of GOD and enhance stability against thermal and urea denaturation [Ahmad et al. (2001) Biochemistry 40, 1947-1955]. In this paper we report the effect of the divalent cations Ca2+ and Mg2+ on the structural and stability properties of GOD. A divalent cation concentration dependent change in native conformation and subunit assembly of GOD was observed. Low concentration (up to 1 M) of CaCl2 or MgCl2 induced compaction of the native conformation of GOD, and the enzyme showed higher stability as compared to the native enzyme against urea denaturation. However, higher concentration (> or =2.0 M) of CaCl2 or MgCl2 induced dissociation of the native dimeric enzyme, resulting in stabilization of the enzyme monomer. An interesting observation was that the 3 M CaCl2-stabilized monomer of GOD retained about 70% secondary structure present in the native GOD dimer; however, there was a complete loss of cooperative interactions between these secondary structural elements present in the enzyme. Regarding the mechanism of divalent cation induced structural changes in GOD, the studies suggest that organization of water molecules by divalent cation results in stabilization of enzyme at low divalent cation concentration, whereas direct binding of these cations to the enzyme, at higher divalent cation concentration, results in dissociation and partial unfolding of the dimeric enzyme molecule.     

GTP cyclohydrolase I utilizes metal-free GTP as its substrate.

GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP. Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.     

Kinetic evidence for a dual cation role for muscle pyruvate kinase

The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg²⁺ and varying concentrations of Mn²⁺ or of Co²⁺. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co²⁺ and varying Mn²⁺. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn²⁺ is a better activator of the enzyme than is Mg²⁺, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni²⁺ or Zn²⁺ with Mg²⁺ suggests that Ni²⁺ and Zn²⁺ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg²⁺. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity.     

Fluorometric studies of aza-epsilon-adenylylated glutamine synthetase from Escherichia coli

Glutamine synthetase in Escherichia coli is regulated by adenylation and deadenylation reactions. The adenylation reaction converts the divalent cation requirement of the enzyme from Mg2+ to Mn2+. Previously, the catalytic action of unadenylated glutamine synthetase was elucidated by monitoring the intrinsic tryptophan fluorescence change accompanying substrate binding. However, due to the lack of changes in the tryptophan fluorescence, a similar study could not be done with the adenylated enzyme. In this study, therefore, an extrinsic fluor is introduced into the adenylated glutamine synthetase by adenylating the enzyme with 2-aza-1,N6-ethenoadenosine triphosphate, a fluorescent analog of ATP. The modified enzyme (aza-epsilon-glutamine synthetase) exhibits catalytic and kinetic properties similar to those of the naturally adenylated enzyme. The results of fluorometric studies on this aza-epsilon-glutamine synthetase indicated that L-glutamate and ATP bind to both Mn2+ and Mg2+ forms of the enzyme in a random order, but only the Mn2+ form is capable of forming a highly reactive enzyme-bound intermediate which is a prerequisite for the reaction with NH4+ to form products. The extrinsic fluorescence changes are also used to determine the binding constants of various substrates and inhibitors of both the biosynthetic and gamma-glutamyl transfer reactions.     

Interaction of Penicillium notatum phospholipase B with divalent cations

The interaction between Penicillium notatum phospholipase B and divalent cations such as Ca2+ and Mg2+ was studied. When the purified enzyme, present at concentrations of submicrogram to microgram per ml, was incubated with submillimolar to millimolar concentrations of CaCl2 or MgCl2, the enzymatic activity was remarkably decreased (to no more than 30% of original activity, when the enzyme was incubated with 2 mM CaCl2 for 15 min). The inhibitory effect of divalent cations was reversible, since dialysis against a metal chelator, such as EDTA or EGTA, substantially restored the enzymatic activity. Atomic absorption analysis showed the purified enzyme molecule to be present in a complex with Ca2+ at a ratio approaching 1:1, and this Ca2+ binding was shown to be extremely tight, since repeated dialyses of the enzyme molecules against EDTA or EGTA could remove the divalent cations only in a gradual manner. During this process, the enzyme activity increased also gradually. The remnant fraction of tightly bound Ca2+ was released from the enzyme molecule after the denaturation of the enzyme by treatment with guanidine hydrochloride, and the apoenzyme recovered its substantial activity after removal of the denaturing agent by dialysis. On the other hand, the content of Mg2+ in the purified enzyme molecule was lower than that of Ca2+, and the association of Mg2+ with the enzyme was much weaker in comparison to that of Ca2+. Atomic absorption analysis of the enzyme exposed to exogenous Ca2+ showed a fast removal, by dialysis, of unbound and weakly bound divalent cation, followed by a gradual removal of endogenous Ca2+ and a concomitant increase of enzymatic activity, which are similar to data obtained for the purified enzyme. Results shown in this report suggest some regulatory roles of divalent cations, especially of Ca2+, in the enzymatic function of P. notatum phospholipse B.     

Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis.

A high yield, photoactivated cross-linking reaction between a modified tRNA and RNase P RNA was used as a quantitative assay of substrate binding affinity. The cross-linking assay allows the effects of metal ions on substrate binding to be measured independently and in the absence of the pre-tRNA cleavage reaction. The results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent cations act primarily to enhance enzyme-substrate binding, presumably by functioning as counterions. This enhancement can be attributed to a reduction in the tRNA off-rate. (ii) Although divalent cation is required for cleavage, the enzyme-substrate complex can form in the absence of divalent cation; the essential role of divalent cation in the reaction is thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but supports catalysis only at a low rate.     

Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.

The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.     

Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.