Quenching of red cell tryptophan fluorescence by mercurial compounds

Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.     

A new class of chromophoric organomercurials and their reactions with d-glyceraldehyde 3-phosphate dehydrogenase

THE SYNTHESES OF THE FOLLOWING ORGANOMERCURIALS ARE DESCRIBED: 2-chloromercuri-4-nitrophenol, 2-chloromercuri-4,6-dinitrophenol, 4-chloromercuri-2-nitrophenol and 2,6-dichloromercuri-4-nitrophenol. All four organomercurials show large spectral changes in the visible spectrum when thiols displace a more weakly bound ligand such as EDTA from the mercury atom. These spectral changes are primarily associated with pK perturbation of the nitrophenols. The mercurials are therefore chromophoric probes for thiol groups in proteins and other thiols of biological interest. The enzyme d-glyceraldehyde 3-phosphate dehydrogenase from lobster muscle is used as a model system in which the properties of the organomercurials may be illustrated. In particular it is shown how d-glyceraldehyde 3-phosphate dehydrogenase carboxymethylated at the active site may be mercurated at a specific site. This mercurial derivative may be crystallized and shown to be isomorphous with the parent enzyme. The mercurials also act as ;reporter groups' by monitoring phosphate or pyrophosphate binding to the enzyme. The mercurials may also be used to estimate cations by an EDTA displacement method.     

Molecular Shape and Packing in Crystals of Soybean β-Amylase

The structure of soybean β-amylase in trigonal (P3₂21) crystals was determined at 4.5 Å resolution by X-ray crystallographic techniques using the isomorphous replacement method. X-Ray diffraction data were collected by the screened precession method for the native enzyme and two heavy atom derivatives. The shape of the enzyme molecule and the locations of mercurial binding are presented. The molecule appeared to be composed of two domains: the larger domain contains one mercurial site on its surface and the smaller domain has another mercurial site, which seemed to be the so-called essential sulfhydryl group. A distinct cleft formed between the domains near the latter sulfhydryl group may be a substrate binding region.     

Mechanisms of inhibition of active transport ATPases by mercurials.

Inhibition by methylmercury and mercuric chloride of Mg,Ca ATPase and Na,K ATPase activities in human erythrocyte ghosts was correlated with the binding capacity of ghosts for the mercurial. Full inhibition was always reached below saturation of binding capacity, and half-inhibition at levels as low as 10% saturation. Under such conditions, concentrations of free inhibitor were negligibly low, and existing mathematical models of inhibition were not applicable. New inhibitor partition equations were introduced to model the mechanisms of action of mercurials. Up to 7 methylmercury groups were calculated to bind to one Na,K ATPase molecule at non-inhibitory sites, while only one reacted with the inhibitory site. Mg,Ca ATPase showed simple one-hit inhibition (one mercurial per enzyme); further washing of ghosts, however, unmasked a second binding site (cooperative two-hit inhibition). Affinities of mercurials to sites of inhibition were calculated relative to other ligands in erythrocyte membranes: the ratios ranged from 3 : 1 to 50 : 1. The results demonstrated the use of binding capacity assays and inhibitor partition equations to measure and compare the susceptibilities of membrane-bound enzymes to poisoning by mercurials.     

Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes.

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.     

Experimental study on the estrogen-like effect of mercuric chloride

Although mercuric chloride has toxicity on reproductive system, it is uncertain if such toxicity is induced by estrogen-like effect. To study whether mercuric chloride has the estrogen-like effect and its relevant mechanism, proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay, peroxidase activity assay and estrogen receptor competitive binding assay were conducted to screen the estrogen-like effect of mercuric chloride. The MCF-7 cells proliferated in the stimulation of mercuric chloride and got to the peak at 10⁻⁷ mol/l concentration. And this proliferation could be completely blocked by estrogenic antagonist ICI182.780. In addition, mercuric chloride could increase the weight of uterus of ovariectomized SD rats and the peroxidase activity of uterus complying with dose-effect relationship. However, mercuric chloride could not affect the binding of estradiol (E₂) to estrogen receptor (ER). So mercuric chloride exhibits the estrogen-like effect through binding and activating ER rather than bind to ER by competing with E₂.     

Differential effects of mercurial compounds on excitable tissues.

Sarcoplasmic reticulum (SR), Ca2+ plus Mg2+-ATPase, and Ca2+-ionophore were obtained from white rabbit skeletal muscles. Methylmercury inhibited the Ca2+ plus Mg2+-ATPase and Ca2+-transport but had no effect on the Ca2+-ionophore. Mercuric chloride inhibited all three functions (i.e., ATPase, transport and ionophoric activity). The mechanism of HgCl2 inhibition of the Ca2+-ionophore was by competition with Ca2+ for Ca2+-ionophoric site whereas its inhibition of the enzyme and Ca2+-transport was due to the blockage of essential sulfhydryl (--SH) groups. Ca2+ plus Mg2+-ATPase and Ca2+-transport were more sensitive to methylmercury than to HgCl2. Acetylcholine receptor (AChR) was obtained for the electric organ of T. californica. Methylmercury inhibited the ACh binding to AChR WITH Ki = 5.7 - 10(-6) M. This effect was not due to mercuric ion alone since mercuric chloride up to 10(-4) M did not affect ACh binding to AChR. It is concluded that: the Ca2+ plus Mg2+-ATPase and Ca2+-transport contain --SH groups essential for their activity, and that the two functions are tightly coupled; the Ca2+-ionophore contains no --SH groups essential for its activity; CH3HgCl inhibition of Ca2+ plus Mg2+-ATPase and Ca2+-transport is partly due to its reactivity with --SH groups in hydrophobic environment; the Ca2+-transport is inhibited by HgCl2 through two processes, one which is the blockage of --SH groups and another which is the inhibition of the Ca2+-ionophoric site; and the inhibition of ACh binding to AChR is due to the blockage of --SH groups in hydrophobic environment, which is inaccessible to Hg2+. Our data present for the first time a molecular basis for the myopathy associated with mercurial compounds toxicity.     

Catalysis of the photochemical dismutation of N-methylacridinium cation to N-methylacridone and N-methyl-9, 10-dihydroacridine by hydrophobic sites of horse-liver alcohol dehydrogenase and human serum albumin.

The N-methylacridinium cation is bound to hydrophobic sites of horse liver alcohol dehydrogenase and human serum albumin with an observed stoichiometry of one molecule N-methyl-acridinium chloride per subunit of alcohol dehydrogenase and 2.5 molecules of the dye per molecule human serum albumin; the dissociation constants are 3.6 X 10(-5) M and 1.7 X 10(-5) M, respectively. In light, the proteins catalyze the dismutation of N-methylacridinium chloride to N-methylacridone and N-methyl-9,10-dihydroacridine. The presence or absence of oxygen has no effect upon the observed reaction rate. If horse liver alcohol dehydrogenase is used as catalyst, the reaction is inhibited by adenosine diphosphoribose and by 1,1'-dimethyl-4,4'-bipyridylium dichloride. It is concluded that the N-methylacridinium cation is bound within the catalytic site of the enzyme interacting with the binding sites of the nicotinium ring and/or the binding site of the lipophilic part of the substrate. The anaerobic photodismutation of N-methylacridinium chloride to N-methyl-9,10-dihydroacridine and N-methylacridone can be explained by several alternative patways (see Appendix by S. Hünig), the overall reaction being 2[N-Methylacridinium]+ + H2Ohw leads to N-Methyl-9,10-dihydroacridine + N-methylacridone + 2H+. The prerequisite, a high rate of proton transfer from the reaction site, seems to be common property of the hydrophobic binding regions for the N-methylacridinium cation in both horse liver alcohol dehydrogenase and human serum albumin.     

Dimerization of papain induced by mercuric chloride and a bifunctional organic mercurial.

The bifunctional mercurial meso-1,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed.     

Resolution of the effects of sulfhydryl-blocking reagents on hormone-and DNA-binding activities of the chick oviduct progesterone receptor

The differential effects of sulfhydryl (SH)-blocking agents on hormone and DNA binding by the chick oviduct progesterone receptor were investigated. Previous studies have demonstrated inhibition of steroid-receptor interaction by SH-blocking agents and protection against inhibition by bound hormone. The present results indicate that the SH group required for steroid binding is within or near the hormone-binding site itself, and that a second SH group (or groups) is involved in the binding of receptor to DNA. Three findings relate to the site of action of SH-blocking agents on hormone binding. First, glycerol decreased the rate of hormone dissociation and the rate of hormone displacement by mercurial reagents by 75 to 90%. Second, mercurial reagents displaced [³H]progesterone bound to the mero-receptor, a M_r 23,000 proteolytic fragment containing the hormone-binding site, but not the site of interaction with DNA. Third, hormone displacement was still present after a 10,000-fold purification of the progesterone receptor. Mercurial reagents also inhibited binding of progesterone receptor to DNA, whereas the SH-alkylating agents N-ethylmaleimide and iodoacetamide had no effect. It is likely that distinct sulfhydryl groups are required for steroid receptor interaction with hormone and with DNA, since brief treatment with mercurial reagents blocked DNA binding, but caused only a slight displacement of bound hormone. The SH group required for hormone binding probably lies within or near the hormone-binding site, is sensitive to mercurials, alkylating agents, and 5,5′-dithiobis(2-nitrobenzoate) (DTNB), and is protected by bound hormone. The SH group required for DNA binding, in contrast, is sensitive to mercurials but not to alkylating agents, is only partially sensitive to DTNB, and is not protected by bound hormone.     

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

The fluorescence and phosphorescence properties of the tryptophan residues in glutamate dehydrogenase were utilized to probe the conformation of the macromolecule at various states of aggregation of its subunits (hexamer, trimer, and monomer) in guanidine hydrochloride. According to the phosphorescence lifetime no gross alteration in the conformation of the protein follows from complete dissociation of the hexamer into native monomer, implying that the native fold is stabilized exclusively by intrasubunit bonding. Although modest concentrations of denaturant induce a change in configuration in the enzyme, a comparison with the macromolecule cross-linked into the hexameric form by glutaraldehyde confirms that this alteration in structure is not the result of subunit dissociation. Inhibition of catalysis by the denaturant is found to be considerably smaller than anticipated from the extent of hexamer dissociation. Furthermore, this inhibition is in no way prevented by cross-linking the enzyme in its hexameric form. This finding together with the ability of the trimer to bind the coenzyme and to undergo the characteristic structural changes induced by the effectors ADP and GTP suggests that, contrary to what is generally believed, the smallest functional unit of glutamate dehydrogenase is not the hexameric form.     

Evaluation of methyl mercury chelating agents using red blood cells and isolated hepatocytes

The relative efficacy of thiol-containing mercurial scavengers was assayed by using cellular suspensions of erythrocytes or isolated hepatocytes. The blood cells incubated in a buffer (pH 7.4) containing 1 mM glucose (10% hematocrit) were exposed to 5 μM methyl mercuric chloride. In the absence of extracellular thiols the red blood cells took up more than 90% of methyl mercury from the surrounding medium during 5–10 min. This uptake was almost completely inhibited by dimercaptosuccinic acid (DMSA) (1 mM) and the same chelant could rapidly remove 80% of the mercury from ‘pre-loaded’ erythrocytes. Hepatocytes prepared according to the method of Seglen [11] in a suspension of 10⁶ cells/ml in a buffer containing 5 mM glucose and 5 mg/ml of bovine serum albumin were also exposed to methyl mercuric chloride (4 μM). Almost 50% of the mercurial was taken up by the cells slowly during the incubation period of 240 min. DMSA (1 mM) almost completely blocked the methyl mercury binding by the hepatocytes. 2-Mercaptopropionylglycin (Thiola) or mercaptosuccinic acid (MSA) was almost as effective mercurial scavengers as DMSA in hepatocytes and in red blood cells. Diethyldithiocarbamate (DDC) and dimercaptopropanol (BAL) were considerably less effective than DMSA to inhibit the mercurial binding to hepatocytes. Experiments in vivo have shown that DMSA is a better mercurial chelator than Thiola or MSA, whereas DDC and BAL may both be considered to be inapplicable in methyl mercury poisonings. Our cellular assay provides preliminary information of the efficiency of chelating thiols and may serve as a useful first approximation when planning further experiments.     

Steroid hormonal regulation of calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats

Ethylenediamine tetraacetate ( EDTA ) inhibits lactoperoxidase (LPO)-catalyzed rate of iodide oxidation in concentration and pH-dependent manner. A plot of log Ki_app values against various pH yields a sigmoidal curve from which an ionisable group of pK_a value 6.0 could be ascertained for controlling the inhibition of catalytically active LPO by EDTA. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation by acting as an electron donor. EDTA al so reduces LPO-compound-11 to the native ferric state by one-electron transfer as evidenced by the spectral shift from 428 to 412 nm. Optical difference spectroscopic studies indicate that EDTA binds to LPO with the apparent equilibrium dissociation constant (KD) of 12 ± 2 mM at pH 6.5. A plot of log K_D values against various pH produces a sigmoidal curve from which an ionisable group of LPO having pk_a = 5.47 could be calculated, deprotonation of which favours EDTA binding. EDTA also binds to LPO-CN- complex indicating its binding site away from heme iron centre. The K_D of LPO-EDTA complex is significantly increased (62 ± 5 mM) by iodide suggesting that EDTA binds close to the iodide binding site. EDTA also increases the K_D value of LPO-hydroquinone complex from 62 ± 5 mM to 200 ± 21 mM indicating that EDTA and aromatic donor binding sites are also close. We suggest that EDTA inhibits iodide oxidation competitively as an electron donor by interacting at or near the iodide binding site and these sites are close to the aromatic donor binding site.     

Rat kidney acylase I: further characterisation and mutation studies on the involvement of Glu 147 in the catalytic process

Rat kidney acylase I was characterised by performing site-directed mutagenesis and enzymatic analysis in the presence of various chemical inhibitors. Site-directed mutagenesis on E147 and overexpression of the protein in a bacterial system, revealed the importance of this residue in enzymatic activity, it corresponds to the putative catalytic E175 in carboxypeptidase G2 from Pseudomonas aeruginosa. The reactivity of histidine and cysteine residues of acylase I with diethylpyrocarbonate (DEPC) and mercuric chloride, respectively, showed that these two amino acids are required for the enzyme to be fully active. Interestingly, the effects of mercuric chloride on rat kidney acylase I were not as great as those on the porcine enzyme, in agreement with previously observed differences between the two enzymes. Moreover, N-[3-(2-furyl)-acryloyl-L-methionine] (FA-Met) a synthetic substrate of the porcine acylase I was found to be an inhibitor of the rat kidney enzyme. These results strongly suggest the existence of differences between the active site of rat and porcine kidney acylases I. Lastly, the rat kidney enzyme was as sensitive as its porcine counterpart to two metal chelating agents, 1,10-phenanthroline and ethylenediamine tetraacetate (EDTA).     

Calcium ion-mediated regulation of the alpha-toxin pore of Staphylococcus aureus.

The water-soluble alpha-toxin monomers of Staphylococcus aureus become hexamers forming the transmembrane pore when exposed to the membranes. This pore is freely permeable to small hydrophilic molecules, e.g. carboxyfluorescein, and becomes less permeable in the presence of calcium ions. Calcium ion-mediated decrease of the carboxyfluorescein leakage could not be eliminated by EDTA added in the medium, but the carboxyfluorescein could be freed by EDTA added in the intraliposomal space. This result suggests that the alpha-toxin pore changes its conformation as the calcium ion is bound and that the binding site is exposed to the intraliposomal side of the membrane. The interaction between the alpha-toxin hexamer and 8-anilino-1-naphthalene-sulfonic acid (ANS) was monitored by determining the fluorescence in the presence and absence of calcium chloride. The mean distances between the tryptophan residues of the alpha-toxin hexamer and the bound ANS were calculated to be 1.90 and 1.80 nm in the absence and presence, respectively, of calcium ions. The results showed the calcium ion mediated conformational change of the membrane-embedded alpha-toxin hexamer.     

35-Cl nuclear magnetic resonance studies of anion-binding sites in proteins: lactate dehydrogenase.

³⁵Cl nmr relaxation rate measurements have been used to study anion-binding sites in pig heart lactate dehydrogenase. These studies reveal two types of sites, one is intimately associated with the active site, the other is not. The nonactive site has been ascribed to a subunit site in analogy with crystallographic results from the dogfish M₄ enzyme. The binding of either the reduced or the oxidized form of NAD results in an increase in the ³⁵Cl nmr relaxation rate by a factor of 1.8–2. The enhanced nmr relaxation rate of the binary lactate dehydrogenase-NAD complex is reduced on binding of the substrate inhibitor molecules oxamate or oxalate to a value less than that exhibited by lactate dehydrogenase alone. The enhancement of the nmr relaxation rate is attributed to a decrease in the dissociation constant of Cl for the enzyme. The K_p values for Cl binding to the active center site of lactate dehydrogenase is 0.85 m and for lactate dehydrogenase-NADH is 0.25 m. The ratio of these constants, 3.4, agrees well with the measured enhancement value 3.7. The effect of coenzyme analogs on the ³⁵Cl nmr relaxation rate has been examined. 3-Acetylpyridine NAD produces an enhancement of 4.3, thionicotinamide NAD of 2.3, whereas 3-pyridinealdehyde, adenosinediphosphoribose, and adenosine diphosphate do not affect the nmr relaxation state of Cl bound to lactate dehydrogenase.     

Molecular origin of the aging effects in glyceraldehyde-3-phosphate dehydrogenase

Rat muscle glyceraldehyde-3-phosphate dehydrogenase was reacted with two reagents aimed at the highly reactive cysteine-149 residue in the active site of the enzyme. The enzyme was rapidly inactivated by iodine monochloride. Complete inactivation occured when approx. 6 mol ICl were added per mol enzyme, indicating that reactions which compete with the reagent's interaction with cysteine-149 take place. Iodine was also found to inactivate the enzyme rapidly and effectively, and, when not in excess, this reagent interacted specifically with cysteine-149. The fraction of original enzymatic activity which could be restored by 2-mercaptoethanol in enzyme samples inhibited by 4.2 mol I₂/mol enzyme, decreased with time to a limiting value of 0.6 reached after approx. 15 min. The enzyme thus treated showed a remarkable similarity to enzyme samples purified from old rats, both in its activity and in NAD⁺ binding patterns under various conditions. It is concluded that the structural modifications induced in the modified enzyme resemble the age-related modifications in native ‘old’ enzyme. These results demonstrate that the origin of the age-related effects in glyceraldehyde-3-phosphate dehydrogenase is in subtle, post-synthetic structural changes. The inactivation reactions described above require a non-reducing environment for the enzyme. Whether such conditions do exist in cells of old animals is the subject of future studies.     

Paradoxical effects of methylmercury on the kinetics of cytochrome c oxidase

A stoichiometric amount of methylmercuric chloride substantially inhibits cytochrome c oxidase function under steady-state turnover conditions, where the enzyme is using its substrates, cytochrome c and oxygen, rapidly and continuously. Under these conditions, a reduction in activity of approximately 40% is observed. This is in accord with the results of Mann and Auer [Mann, A.J., & Auer, H.E. (1980) J. Biol. Chem. 255, 454-458], who used mercuric chloride and ethylmercuric chloride. Paradoxically, we found that addition of methylmercuric chloride can increase the activity of cytochrome c oxidase during its initial substrate utilization. This rate enhancement, measured under conditions where the enzyme cycles only a few times, is maximal for the resting state of the enzyme. "Pulsed" cytochrome c oxidase (i.e., enzyme that has been recently reduced and reoxidized) is considerably activated with respect to the resting enzyme, showing faster turnover rates (Antonini, 1977; Brunori et al., 1979). No significant rate enhancement upon treatment with methylmercuric chloride is seen in initial substrate utilization if the enzyme is pulsed immediately before the assay. The apparently contradictory effects of methylmercuric chloride on the resting and pulsed states of the oxidase under low turnover conditions may be reconciled by a model in which mercurial binding greatly stabilizes the enzyme in a state resembling that of the pulsed enzyme. A decrease in conformational flexibility may be the basis of the mercurial-induced diminution in activity of the enzyme during steady-state turnover conditions.     

Biomonitoring of low levels of mercurial derivatives in water and soil by Allium micronucleus assay

The Allium micronucleus (MNC) assay was developed to monitor low levels of mercury in aquatic and terrestrial environments. Four mercurial derivatives namely mercuric chloride (MC), methyl mercuric chloride (MMC), phenyl mercuric acetate (PMA) and a methoxy ethyl mercuric chloride based fungicide, Emisan-6, were tested to assess the sensitivity and versatility of the Allium MNC assay. Allium bulbs were set directly on water and soil contaminated with known levels of mercurial derivatives (0.0001-10.00 ppm). On the 5th day the endpoints measured were root length, mitoses with spindle abnormality and cells with MNC in root meristems. The effective concentrations of the test chemicals that cause 50% of root length as compared to control (EC50) were determined from dose-response curves so obtained. The lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for each of the mercurial derivatives for the induction of spindle malfunction and MNC were determined. It was found that EC50, LECT and HICT values for mercurial derivatives in soil were higher than those in water. The frequencies of cells with MNC and mitoses with spindle abnormality were highly correlated indicating that MNC is a good parameter of spindle malfunction. The present approach increased the sensitivity of the Allium assay by 10-fold, the detection limit being 0.001-0.1 ppm and 0.1-1.0 ppm in aquatic and terrestrial environments respectively, depending on the species of mercury.     

Structural basis of substrate selectivity of Δ-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): Semialdehyde chain length

The enzyme Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase (aka P5CDH and ALDH4A1) is an aldehyde dehydrogenase that catalyzes the oxidation of γ-glutamate semialdehyde to l-glutamate. The crystal structures of mouse P5CDH complexed with glutarate, succinate, malonate, glyoxylate, and acetate are reported. The structures are used to build a structure-activity relationship that describes the semialdehyde carbon chain length and the position of the aldehyde group in relation to the cysteine nucleophile and oxyanion hole. Efficient 4- and 5-carbon substrates share the common feature of being long enough to span the distance between the anchor loop at the bottom of the active site and the oxyanion hole at the top of the active site. The inactive 2- and 3-carbon semialdehydes bind the anchor loop but are too short to reach the oxyanion hole. Inhibition of P5CDH by glyoxylate, malonate, succinate, glutarate, and l-glutamate is also examined. The K_i values are 0.27 mM for glyoxylate, 58 mM for succinate, 30 mM for glutarate, and 12 mM for l-glutamate. Curiously, malonate is not an inhibitor. The trends in K_i likely reflect a trade-off between the penalty for desolvating the carboxylates of the free inhibitor and the number of compensating hydrogen bonds formed in the enzyme-inhibitor complex.