Binding Affinity Of Enzyme Research Articles

CDNA Cloning, Protein Structure Modeling of Rhesus Monkey ( Macaca mulatta) Prothrombin

Objective Hemorrhagic diseases have been considered to be one of the main causes of xenotransplantation failure. To explore the role of the rhesus monkey ( Macaca mulatta) coagulation system in a pig-to-human xenotransplantation model, we primarily studied the full-length cDNA and the three-dimensional (3-D) structures of the important coagulation factor-prothrombin of the rhesus. Materials and Methods The full-length cDNA of rhesus prothrombin was obtained by rhesus monkey liver cDNA library screening and a 5′RACE technique. The 3-D protein structure was modeled using the SWISS-MODEL program. The important macromolecular interaction sites were compared with human and porcine prothrombin. Results At first, the full-length rhesus prothrombin cDNA was cloned; the sequence was submitted to the Genebank (accession number: EF057490). The full-length cDNA is 2029 bp, with a complete open reading frame of 1884 bp, coding 626 amino acids. The deduced protein sequence contains a signal peptide, propeptide, Gla domain, two Kringle domains, and a Trypsin domain. The nucleotide similarities of rhesus-human and rhesus-porcine genes are 95.64% and 86.14%, and those of the amino acids, 94.51% and 82.82%, respectively. Some important functional sites, such as the catalytic triad DHS, RGD, Na +-binding, and the carboxylase-binding site, are highly conserved. However, among the three species some variations are observed in potential N-glycosylation, O-glycosylation, phosphorylation, cleavage site, FXa-binding sites. Especially, the autolysis loop shows great differences in both amino acid sequence and 3-D model. Conclusions The great similarity of prothrombin among rhesus, human, and porcine confirmed the great value of the pig-to-rhesus xenotransplantation model. The variation especially in some important recognition sites between rhesus and pig would vary the binding affinity of enzymes to xenosubstrates and their reaction velocities in postoperative coagulation processes, which would be one possible reason for the disordered regulation of clotting seen during the xenorejection in animal models.

Stoichiometric Selection of Tight-Binding Inhibitors by Wild-Type and Mutant Forms of Malarial (Plasmodiumfalciparum) Dihydrofolate Reductase

A simple method for screening combinatorial and other libraries of inhibitors of malarial (Plasmodium falciparum) dihydrofolate reductase (PfDHFR) has been developed, based on the affinities of the inhibitors with the enzyme. In the presence of limiting amounts of the enzyme, a number of inhibitors in the library were bound to extents reflecting the relative binding affinities. Following ultrafiltration and guanidine hydrochloride treatment to release bound inhibitors, the amounts of free and bound inhibitors could be determined by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The differences in the patterns reflected the binding of high-affinity components compared with the other members in the library. A good correlation was found between the inhibition constants (Ki values) and the extent of binding of inhibitors to wild-type, double (C59R+S108N) and quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well as human DHFR. In addition to identifying lead components of the libraries with high affinities (low Ki values) and stabilities (low k(off) rates), this simple method also provides an alternative way for quickly and accurately calculating enzyme binding affinities of inhibitors in combinatorial chemical libraries.

Delaying the Mitochondrial Decay of Aging with Acetylcarnitine

Oxidative mitochondrial decay is a major contributor to aging. Some of this decay can be reversed in old rats by feeding them normal mitochondrial metabolites, acetylcarnitine (ALC) and lipoic acid (LA), at high levels. Feeding the substrate ALC with LA, a mitochondrial antioxidant, restores the velocity of the reaction (K(m)) for ALC transferase and mitochondrial function. The principle appears to be that, with age, increased oxidative damage to protein causes a deformation of structure of key enzymes with a consequent lessening of affinity (K(m)) for the enzyme substrate. The effect of age on the enzyme-binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product that increases with age). In old rats (vs. young rats), mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O(2) uptake are lower; oxidants/O(2), neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher. Ambulatory activity and cognition decline with age. Feeding old rats ALC with LA for a few weeks restores mitochondrial function; lowers oxidants, neuron RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (as assayed with the Skinner box and Morris water maze). A recent meta-analysis of 21 double-blind clinical trials of ALC in the treatment of mild cognitive impairment and mild Alzheimer's disease showed significant efficacy vs. placebo. A meta-analysis of 4 clinical trials of LA for treatment of neuropathic deficits in diabetes showed significant efficacy vs. placebo.

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Novel P4 truncated tripeptidyl α-ketoamides as HCV protease inhibitors

We describe herein the synthesis and evaluation of two series of P-4 truncated tripeptidyl α-ketoamides as HCV serine protease inhibitors. The most promising compound disclosed in this communication 7b demonstrated enzyme binding affinity ( K i) at 0.27 uM.

1,2,4-Thiadiazole: A novel Cathepsin B inhibitor

A novel class of Cathepsin B inhibitors has been developed with a 1,2,4-thiadiazole heterocycle as the thiol trapping pharmacophore. Several compounds with different dipeptide recognition sequence (i.e., P1′– P2′=Leu-Pro-OH or P2– P1=Cbz-Phe-Ala) at the C5 position and with different substituents (i.e., OMe, Ph, or COOH) at the C3 position of the 1,2,4-thiadiazole ring have been synthesized and tested for their inhibitory activities. The substituted thiadiazoles 3a– h inhibit Cat B in a time dependent, irreversible manner. A mechanism based on active-site directed inactivation of the enzyme by disulfide bond formation between the active site cysteine thiol and the sulfur atom of the heterocycle is proposed. Compound 3a ( K i=2.6 μM, k i K i =5630 M −1 s −1 ) with a C3 methoxy moiety and a Leu-Pro-OH dipeptide recognition sequence, is found to be the most potent inhibitor in this series. The enhanced inhibitory potency of 3a is a consequence of its increased enzyme binding affinity (lower K i) rather than its increased intrinsic reactivity (higher k i). In addition, 3a is inactive against Cathepsin S, is a poor inhibitor of Cathepsin H and is >100-fold more selective for Cat B over papain.

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.

Electrocyte (Na +,K +)ATPase inhibition induced by zinc is reverted by dithiothreitol

The Mg 2+-dependent (Na +,K +)ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn 2+) on the (Na +,K +)ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn 2+ is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na +,K +)ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na +,K +)ATPase from both membrane fractions and was able to revert the Zn 2+ inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na +,K +)ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na +,K +)ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn–dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn 2+ may be considered in regard to its neurotoxic effects.

Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans.

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.

Computational studies on HIV-1 protease inhibitors: influence of calculated inhibitor-enzyme binding affinities on the statistical quality of 3D-QSAR CoMFA models.

A theoretical study was performed on a set of 38 human immunodeficiency type 1 (HIV-1) protease inhibitors that are structurally similar to the AIDS drug Indinavir. Comparison between the computed binding energies and experimental activity data (pIC(50)) found a high degree of correlation (r(2)() = 0.82). Three-dimensional quantitative structure-activity relationship (3D-QSAR) models using comparative molecular field analysis (CoMFA) yielded predicted activities that were in excellent agreement with the corresponding experimentally determined values. Inclusion of the calculated enzyme-inhibitor binding energy as an additional descriptor in the CoMFA model yielded a significant improvement in the internal predictive ability of our model (q(2)() = 0.45 to q(2)() = 0.69). Separate CoMFA models were constructed to evaluate the influence of different alignment schemes (Atom Fit and Field Fit) and different partial atomic charge assignment schemes (Discover CVFF, Gasteiger-Marsili, and AM1-ESP) on the statistical quality of the models.

Phosphorylation of calmodulin alters its potency as an activator of target enzymes.

Previous work has shown that calmodulin (CaM) is constitutively phosphorylated in rat liver, probably by casein kinase II [Quadroni, M., James, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A procedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation experiments yield a 50:50 mixture of 3-4 times phosphorylated CaM and native CaM. The activation of six target enzymes by PCaM was tested: myosin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In general, the phosphorylation of CaM caused a decrease in enzyme binding affinity, increasing the Kact by 2-4-fold for MLCK, PDE, PM Ca2+-ATPase, and calcineurin. The Vmax at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only minimally activated by PCaM and NOS whose Vmax was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very little effect on the binding of calcium to the enzyme despite the fact that Ser 101 which is phosphorylated is located in the third calcium binding loop. CD measurements performed on CaM and PCaM indicated that phosphorylation causes a marked decrease in the alpha-helical content of the protein. Phosphorylated CaM is very prone to dephosphorylation and was thus tested as a substrate for several phosphatases. It was unaffected by calcineurin (PP2B), but was a reasonable substrate for the pleiotropic phosphatases PP1gamma and PP2A.

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent Km ∼ 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (Vmax, 895 fmol/h/mg protein) than the female (Vmax, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5α-reduced metabolite of testosterone, 5α-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (Ki = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.

Promoter Selectivity of Escherichia coli RNA Polymerase Eσ70 and Eσ38 Holoenzymes: EFFECT OF DNA SUPERCOILING

The functional specificity was compared between two sigma factors, sigma 70 (the major sigma at exponentially growing phase) and sigma 38 (the essential sigma at stationary growth phase), of Escherichia coli RNA polymerase. The core enzyme binding affinity of sigma 38 was less than half the level of sigma 70 as measured by gel filtration column chromatography or by titrating the concentration of sigma required for the maximum transcription in the presence of a fixed amount of core enzyme. In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for E sigma 38 than E sigma 70. The transcription by E sigma 38 was, however, enhanced with the use of templates with low superhelical density, in good agreement with the decrease in DNA superhelicity in the stationary growth phase. We thus propose that the selective transcription of stationary-specific genes by E sigma 38 holoenzyme requires either a specific reaction condition(s) or a specific factor(s) such as template DNA with low superhelical density.

Escherichia coli DNA topoisomerase III is a site-specific DNA binding protein that binds asymmetrically to its cleavage site.

The binding of DNA topoisomerase III (Topo III) to a single-stranded DNA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases; 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indicate that the enzyme also binds to its substrate asymmetrically, protecting approximately 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomerase. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by Topo III than an oligonucleotide without a cleavage site.

Opposite stereospecificity of two tropinone reductases is conferred by the substrate-binding sites.

Two tropinone reductases (TRs) catalyze opposite stereospecific reductions at a branching point in the biosynthetic pathway of tropane alkaloids. The two TRs, TR-I and TR-II, reduce the 3-keto group of the common substrate tropinone stereospecifically to 3 alpha- and 3 beta-hydroxy groups, to produce the stereoisomeric alkamines tropine and pseudotropine, respectively. Sixteen chimeric TR enzymes were expressed in Escherichia coli, and their stereospecificities, substrate specificities, and Km values for tropinone were compared with those of the wild-type enzymes. Stereospecificity and substrate specificity of the chimeric enzymes were closely correlated, and the carboxyl-terminal peptides of about 120 amino acid residues, in which 53 residues were different between TR-I and TR-II, were shown to determine both specificities. Further dissection of these peptide segments resulted in either enzymes with both TR activities or inactive enzymes. The substrate binding affinity of many chimeric enzymes was much lower than that of wild-type enzymes. These results indicate that the stereospecificity of TR is determined by the orientation of tropinone at the substrate-binding site, which is composed mainly of the carboxyl-terminal half region, and also that the amino-terminal half region constitutes the NADPH-binding site as postulated for short chain nonmetal dehydrogenases.

Inhibitors of the protease from human immunodeficiency virus: synthesis, enzyme inhibition, and antiviral activity of a series of compounds containing the dihydroxyethylene transition-state isostere.

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N- and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective 1(S)-amino-2(R)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-1/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

A higher affinity of AMV reverse transcriptase for template-primers correlates with a lower rate of DNA synthesis

The kinetics of copying of poly (A)-oligo (dT) and poly (C)-oligo (dG) by reverse transcriptase from avian myeloblastosis virus have been studied, and binding affinity of enzyme for template-primer and primer alone have been determined separately. Although the maximal rate of DNA synthesis obtained with poly (C)-oligo (dG) is higher than that for poly (A)-oligo (dT), the binding affinity of the enzyme for poly (C)-oligo (dG) or oligo (dG) is considerably lower than that for poly (A)-oligo (dT) or oligo (dT). Hence, for the more efficient template, poly (C)-oligo (dG), both template-primer and primer bind less tightly to the enzyme.

Bacterial Luciferase: CHEMISTRY OF THE REACTIVE SULFHYDRYL

Abstract Bacterial luciferase catalyzes the bioluminescent oxidation of FMNH2 and a long chain aliphatic aldehyde by O2, producing FMN and the corresponding carboxylic acid. The enzyme from bacterial strain MAV has been found to be inactivated by modification of a single cysteinyl residue per enzyme molecule by alkylating reagents such as iodoacetamide, iodoacetate, and N-ethylmaleimide. The apparent second order rate constants for inactivation at pH 7.0 (25°) are 1.67 x 103 m-1 min-1 for N-ethylmaleimide and 7.0 m-1 min-1 for iodoacetamide. Inactivation by iodoacetate at pH 7.0 is much less rapid, and the rate constant was not determined below pH 8. With all three reagents, the rate of inactivation increases with increasing pH in a manner suggesting a pKa of about 9.4 for the reactive sulfhydryl group. The rate of inactivation by N-[1-14C]ethylmaleimide correlates with the rate of modification of one sulfhydryl group on the α subunit, the β subunit being modified much less rapidly. Long chain aliphatic aldehydes protect the enzyme against inactivation by N-ethylmaleimide, the degree of protection increasing with increasing aldehyde chain length. The reactive sulfhydryl is also protected by FMN against alkylation. No binding of FMNH2 could be measured for iodoacetamide- or N-ethylmaleimide-modified enzyme. These results suggest that the reactive sulfhydryl is in or near the active center of the enzyme. The pH profiles of FMNH2 binding affinity and several catalytic parameters of the native enzyme do not implicate a group with a pKa near that of the sulfhydryl. The catalytic function (if any) for the reactive cysteine remains unknown.

The Binding of Reduced Nicotinamide Adenine Dinucleotide Phosphate to Mammalian and Avian Fatty Acid Synthetases: NUMBER OF BINDING SITES AND THE EFFECT OF REAGENTS AND CONDITIONS ON THE BINDING OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE TO ENZYME

Abstract The binding of NADPH to pigeon and rat liver fatty acid synthetases has been examined by the techniques of fluorescence enhancement, fluorescence polarization, and equilibrium dialysis. Pigeon and rat liver fatty acid synthetases increase the fluorescent yield from NADPH 4.6- and 3.8-fold, respectively. Fluorescent yield appears to be independent of the buffer concentration and the amount of coenzyme bound to enzyme. The number of binding sites on each synthetase is independent of the protein concentration. The pigeon liver enzyme binds 2 moles of NADPH per mole of enzyme in 0.2 m phosphate and 3 moles of coenzyme at lower buffer concentrations. The binding affinity for NADPH is independent of the quantity of coenzyme bound; however, binding of coenzyme is competitive with charged species supplied by the buffer. The Kd value in 0.2 m phosphate is 8 µm, and in 0.1 m phosphate it is 3.5 µm. The rat enzyme binds maximally 4 moles of NADPH per mole of enzyme in 0.2 m and in lower buffer concentrations. This enzyme has either two pairs of binding sites or it exhibits negative cooperativity. The affinity of binding of NADPH to each of these pairs of sites is dependent upon the buffer concentration. The dissociation constant for the stronger pair of binding sites is 1 µm in 0.2 m phosphate and less than 1 µm at lower buffer concentrations. Neither the binding of NADPH to protein nor enzyme activity for fatty acid synthesis requires phosphate. Fatty acid synthesis proceeds most rapidly at ionic strengths higher than those yielding the strongest NADPH binding. The binding affinity of the pigeon liver enzyme for NADPH is not changed by the binding of either acetate or malonate (from their respective CoA esters) to protein, thereby indicating a separate binding site for NADPH. It is not affected either by a pH change in the region where the rate of fatty acid synthesis changes rapidly. It is also not changed appreciably by dissociation (with Tris-glycine buffer) of the enzyme complex into half-molecular weight units, a condition that completely eliminates fatty acid synthesis. The binding capacity of the enzyme for NADPH is destroyed by extensive sulfhydryl oxidation and by alkylation with iodoacetamide. It is suggested that the binding of NADPH to enzyme is primarily electrostatic and that it is dependent on one or more sulfhydryl groups. It is not, however, dependent on the noncovalent binding forces responsible for maintaining the integrity of the enzyme complex.