A bioluminescent assay for measuring glucose uptake.

Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts.

A bioluminescence assay for aldehyde dehydrogenase activity.

The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75.

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

Characterization of active site residues of nitroalkane oxidase.

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.

Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction.

The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. In particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution.

Crystal structures of intermediates in the nitroalkane oxidase reaction.

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 A resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes [Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 125, 8738-8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped [Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066]. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.

Bioluminescent assays for ADMET.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.

Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase.

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.

A bioluminescent assay for monoamine oxidase activity.

This article describes a novel two-step homogeneous bioluminescent assay for monoamine oxidase (MAO) that is simple, sensitive, and amenable to high-throughput screening. In the first step, MAO reacts with an aminopropylether analog of methyl ester luciferin. In the second step, a luciferin detection reagent inactivates MAO and converts the product of the first step into a luminescent signal. The amount of light produced is proportional to the amount of MAO and the time of incubation in the first step, but the luminescent signal is stable in the second step with a half-life greater than 5h. The assay has high precision, is more sensitive than current fluorescent methods, and can accurately measure the binding constants of known substrates and inhibitors. An automated screen of the Sigma-RBI Library of Pharmacologically Active Compounds (LOPAC(1280)) revealed a surprisingly high percentage of MAO inhibitors (16%) with a low false hit rate (0.9%). This implies that a significant number of compounds interact with the MAO enzymes and suggests that it is important to include MAO assays in drug metabolism studies. Other advantages of this bioluminescent assay over comparable fluorescent assays are discussed.

New bioluminogenic substrates for monoamine oxidase assays.

Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective beta-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.

Crystal structures of nitroalkane oxidase: insights into the reaction mechanism from a covalent complex of the flavoenzyme trapped during turnover.

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We present the 2.2 A resolution crystal structure of NAO trapped during the turnover of nitroethane as a covalent N5-FAD adduct (ES*). The homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites in an orthorhombic space group. The electron density for the N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The structure of ES was used to solve the crystal structure of oxidized NAO at 2.07 A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A, and there are six subunits (1(1)/(2) holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E(ox)). The active-site structures of E(ox), EI, and ES* reveal a hydrophobic channel that extends from the exterior of the protein and terminates at Asp402 and the N5 position on the re face of the FAD. Thus, Asp402 is in the correct position to serve as the active-site base, where it is proposed to abstract the alpha proton from neutral nitroalkane substrates. The structures for NAO and various members of the ACAD family overlay with root-mean-square deviations between 1.7 and 3.1 A. The homologous region typically spans more than 325 residues and includes Glu376, which is the active-site base in the prototypical member of the ACAD family. However, NAO and the ACADs exhibit differences in hydrogen-bonding patterns between the respective active-site base, substrate molecules, and FAD. These likely differentiate NAO from the homologues and, consequently, are proposed to result in the unique reaction mechanism of NAO.

Prevalence of severe pelvic organ prolapse in relation to job description and socioeconomic status: a multicenter cross-sectional study.

The aim of this study was to determine if certain occupations or socioeconomic levels are associated with pelvic organ prolapse. Investigators at six American sites performed pelvic organ prolapse quantification examinations on women presenting for routine gynecologic care. Between September 1999 and March 2002, 1,004 patients were examined. Severe pelvic organ prolapse was defined as the leading edge being 1 cm or more beyond the hymeneal ring. The data was analyzed with the Kruskal-Wallis analysis of variance, Bonferroni test, multiple logistic regression, and descriptive statistics. The prevalence of severe pelvic organ prolapse in our group was 4.3%. Women who were laborers/factory workers had significantly more severe prolapse than the other job categories (p < 0.001). Women with annual income of Dollars 10,000 or less had significantly more severe pelvic organ prolapse than other income groups (p < 0.001). These differences persisted even when controlling for age, race, number of deliveries, body mass index >30, and smoking status (all p < 0.001). Laborers/factory worker jobs and an annual household income of Dollars 10,000 or less are associated with severe pelvic organ prolapse.

Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis.

The active site Fe(III) of protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH(-). Tyr447 and OH(-) are displaced as protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O(2) becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes.

The effect of pregnancy and mode of delivery on the prevalence of urinary and fecal incontinence.

OBJECTIVE: The purpose of this study was to determine the relative effects of pregnancy and mode of delivery on the prevalence of urinary and fecal incontinence. STUDY DESIGN: This was a prospective, observational multicenter study of women presenting to 6 gynecology clinics. Demographic data collected included: height, weight, gravidity, parity, and number of vaginal deliveries. Patients were diagnosed with incontinence by questionnaire. Standard univariate logistic regression analyses' were performed to determine the contribution of pregnancy, mode of delivery, and BMI on the prevalence of urinary and fecal incontinence. RESULTS: One thousand and four women were enrolled over an 18-month period. Two hundred and thirty-seven and 128 subjects had urinary and fecal incontinence, respectively. Odds ratio (95% CI) calculated for the prevalence of urinary incontinence by pregnancy and mode of delivery were: any term pregnancy vs no term pregnancy was 2.46 (1.53-3.95), any term pregnancy but no vaginal deliveries (cesarean section only) vs no term pregnancy was 1.95 (0.99-3.80), any term pregnancy and at least 1 vaginal delivery vs no term pregnancy was 2.53 (1.57-4.07), and any term pregnancy but no vaginal delivery (cesarean section only) vs any term pregnancy, and at least 1 vaginal delivery was 1.30 (0.77-3.95). Odds ratio (95% CI) calculated for the prevalence of fecal incontinence by pregnancy and mode of delivery were: any term pregnancy vs no term pregnancy was 2.26 (1.22-4.19), any term pregnancy but no vaginal deliveries (cesarean section only) vs no term pregnancy was 1.13 (0.43-2.96), any term pregnancy and at least 1 vaginal delivery vs no term pregnancy was 2.41 (1.30-4.49), and any term pregnancy but no vaginal deliveries (cesarean section only) vs any term pregnancy, and at least 1 vaginal delivery was 2.15 (0.97-4.77). BMI and age did not impact these results. CONCLUSION: Pregnancy increases the risk of urinary and fecal incontinence. Cesarean section does not decrease the risk of urinary or fecal incontinence compared to pregnancy with a vaginal delivery.

Pelvic Organ Support Study (POSST) and bowel symptoms: straining at stool is associated with perineal and anterior vaginal descent in a general gynecologic population.

OBJECTIVE: The purpose of this study was to evaluate the association of constipation symptoms and anal incontinence with vaginal wall and pelvic organ descent in a general gynecologic population. STUDY DESIGN: In this multicenter, cross-sectional study, 1004 women attending routine gynecologic healthcare underwent pelvic organ prolapse quantification (POPQ) measurements, and were surveyed regarding anal incontinence, digitation, < 2 bowel movements (BMs)/week, and > 25% frequency of: straining, hard/lumpy stools, and incomplete emptying. Constipation scores reflected the sum of positive responses. Associations between POPQ measurements (Ba, C, Bp, gh+pb), constipation scores, and anal incontinence were evaluated using multivariable regression. RESULTS: Of 119 women with Bp > or = -1.00, 47% reported no constipation symptoms. Hard/lumpy stools (26%), incomplete emptying (24%), and straining (24%) were more prevalent; fewer women reported < 2 BMs/week (15%) or digitation (7%). Constipation scores were weakly correlated with Bp, gh+pb (both r < .1, P < .02). Women reporting > or = 2 symptoms had greater gh+pb measurements than women reporting 0 or 1 symptom (P = .03). Women with anal incontinence had greater gh+pb and gh values than women without anal incontinence (P < .01). POPQ measurements were regressed separately onto (1) total constipation scores, (2) dichotomized scores, and (3) individual symptoms, with BMI, age, number of vaginal deliveries (NVD), weight of largest vaginal delivery (WLVD), race, hysterectomy, study site, and income included as covariates. Total constipation scores and dichotomized scores were nonsignificant in all models. With regard to individual symptoms, straining at stool was significant in the models for Ba and gh+pb, with greater Ba and gh+pb measurements among strainers relative to nonstrainers. CONCLUSION: Most associations between bowel symptoms and vaginal or pelvic organ descent were weak. After controlling for important covariates, straining at stool remained associated with anterior vaginal wall and perineal descent.

Pelvic Organ Support Study (POSST): the distribution, clinical definition, and epidemiologic condition of pelvic organ support defects.

OBJECTIVE: The purpose of this study was to describe the distribution of pelvic organ support in a gynecologic clinic population to define the clinical disease state of pelvic organ prolapse and to analyze its epidemiologic condition. STUDY DESIGN: This was a multicenter observational study. Subjects who were seen at outpatient gynecology clinics who required an annual gynecologic examination underwent a pelvic organ prolapse quantification examination and completed a prolapse symptom questionnaire. Receiver operator characteristic curves were used to define pelvic organ prolapse with the use of symptoms and pelvic organ prolapse quantification examination measures. Standard age-adjusted univariate and multivariate logistic regression analysis were used to evaluate various relationships. RESULTS: The population consisted of 1004 women who were aged 18 to 83 years. The prevalence of pelvic organ prolapse quantification stages was 24% (stage 0), 38% (stage 1), 35% (stage 2), and 2% (stage 3). The definition of pelvic organ prolapse that was determined by the receiver operator characteristic curve was the leading edge of their vaginal wall that was -0.5 cm above the hymenal remnants. Multivariate analysis revealed age, Hispanic race, increasing body mass index, and the increasing weight of the vaginally delivered fetus as risk factors for pelvic organ prolapse, as defined in this population. CONCLUSION: The results from this population suggest that there is a bell-shaped distribution of pelvic organ support in a gynecologic clinic population. Advancing age, Hispanic race, increasing body mass index, and the increasing weight of the vaginally delivered fetus have the strongest correlations with prolapse.

Establishing the kinetic competency of the cationic imine intermediate in nitroalkane oxidase.

The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2-(2H2)-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent with cyanide trapping this electrophilic intermediate.

Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase.

While several flavoproteins will oxidize nitroalkanes in addition to their physiological substrates, nitroalkane oxidase (NAO) is the only one which does not require the anionic nitroalkane. This, in addition to the induction of NAO by nitroethane seen in Fusarium oxysporum, suggests that oxidation of a nitroaliphatic species is the physiological role of the enzyme. Mechanistic studies of the reaction with nitroethane as substrate have established many of the details of the enzymatic reaction. The enzyme is unique in being the only flavoprotein to date for which a carbanion is definitively established as an intermediate in catalysis. Recent structural analyses show that NAO is homologous to the acyl-CoA dehydrogenase and acyl-CoA oxidase families of enzymes. In NAO, the glutamate which acts as the active site base in the latter enzymes is replaced by an aspartate.