Human glutathione-dependent formaldehyde dehydrogenase. Structures of apo,binary, and inhibitory ternary complexes

The human glutathione-dependent formaldehyde dehydrogenase is unique among the structurally studied members of the alcohol dehydrogenase family in that it follows a random bi bi kinetic mechanism. The structures of an apo form of the enzyme, a binary complex with substrate 12-hydroxydodecanoic acid, and a ternary complex with NAD+ and the inhibitor dodecanoic acid were determined at 2.0, 2.3, and 2.3 A resolution by X-ray crystallography using the anomalous diffraction signal of zinc. The structures of the enzyme and its binary complex with the primary alcohol substrate, 12-hydroxydodecanoic acid, and the previously reported binary complex with the coenzyme show that the binding of the first substrate (alcohol or coenzyme) causes only minor changes to the overall structure of the enzyme. This is consistent with the random mechanism of the enzyme where either of the substrates binds to the free enzyme. The catalytic-domain position in these structures is intermediate to the "closed" and "open" conformations observed in class I alcohol dehydrogenases. More importantly, two different tetrahedral coordination environments of the active site zinc are observed in these structures. In the apoenzyme, the active site zinc is coordinated to Cys44, His66 and Cys173, and a water molecule. In the inhibitor complex, the coordination environment involves Glu67 instead of the solvent water molecule. The coordination environment involving Glu67 as the fourth ligand likely represents an intermediate step during ligand exchange at the active site zinc. These observations provide new insight into metal-assisted catalysis and substrate binding in glutathione-dependent formaldehyde dehydrogenase.     

Multivalent binding of nonnative substrate proteins by the chaperonin GroEL

The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.     

Ligand binding and protein dynamics in lactate dehydrogenase

Recent experimental studies suggest that lactate dehydrogenase (LDH) binds its substrate via the formation of a LDH/NADH.substrate encounter complex through a select-fit mechanism, whereby only a minority population of LDH/NADH is binding-competent. In this study, we perform molecular dynamics calculations to explore the variations in structure accessible to the binary complex with a focus on identifying structures that seem likely to be binding-competent and which are in accord with the known experimental characterization of forming binding-competent species. We find that LDH/NADH samples quite a range of protein conformations within our 2.148 ns calculations, some of which yield quite facile access of solvent to the active site. The results suggest that the mobile loop of LDH is perhaps just partially open in these conformations and that multiple open conformations, yielding multiple binding pathways, are likely. These open conformations do not require large-scale unfolding/melting of the binary complex. Rather, open versus closed conformations are due to subtle protein and water rearrangements. Nevertheless, the large heat capacity change observed between binding-competent and binding-incompetent can be explained by changes in solvation and an internal rearrangement of hydrogen bonds. We speculate that such a strategy for binding may be necessary to get a ligand efficiently to a binding pocket that is located fairly deep within the protein's interior.     

Control of L-ornithine specificity in Escherichia coli ornithine transcarbamoylase. Site-directed mutagenic and pH studies

Escherichia coli ornithine transcarbamoylase displays a strict specificity toward its second substrate L-ornithine. After forming a binary complex with carbamoyl phosphate and undergoing an induced-fit isomerization (Miller, A. W., and Kuo, L. C. (1990) J. Biol. Chem. 265, 15023-15027), the enzyme selects only the minor, zwitterionic ornithine with an uncharged delta-amino group for transcarbamoylation. Formation of the productive ternary complex is linked to two enzymic ionizations (pK alpha 6.2 approximately 6.3 and 9.1 approximately 9.3) and two ornithine ionizations (pK alpha 8.5 and 10.6) (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761). To elucidate the mechanism through which substrate specificity is achieved, the binding of L-ornithine to two site-specific point mutants (Arg-57----Gly and Cys-273----Ala) of the enzyme has been examined. For the Gly-57 mutant enzyme, which does not undergo the induced-fit isomerization, affinity for ornithine drops by a factor of 500. The pH profile of the apparent equilibrium constant governing the association of L-ornithine to the binary complex of this mutant reveals that only two enzymic ionizations affect ornithine binding. The ionizations linked to L-ornithine are not detected. Hence, the preisomerized binary complex binds not only poorly but also indiscriminately all ionic species of L-ornithine. For the Ala-273 mutant enzyme, which exhibits the induced-fit isomerization, affinity of the amino acid is decreased by an order of magnitude. Ionizations of L-ornithine to yield a zwitterion for binding are detected in pH analyses for this mutant, but the pK alpha of 6.2 associated with the enzymic deprotonation in the wild type is absent. Therefore, Cys-273 is a binding site of L-ornithine. The D-isomer of ornithine is a very weak, deadend ligand to all three forms of the enzyme with affinities in the millimolar range. Employing the estimated affinities of D- and L-ornithine, the binding stereospecificity of the wild-type and mutant binary complexes toward the amino acid substrate may be evaluated. L-Ornithine binds preferentially over D-ornithine by two and four orders of magnitude in the absence and presence of protein isomerization, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bacteriophage T4 Dam DNA-[N6-adenine]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.     

Structural and Dynamic Features of the MutT Protein in the Recognition of Nucleotides with the Mutagenic 8-Oxoguanine Base

Escherichia coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, an event that can prevent the misincorporation of 8-oxoguanine opposite adenine in DNA. Of the several enzymes that recognize 8-oxoguanine, MutT exhibits high substrate specificity for 8-oxoguanine nucleotides; however, the structural basis for this specificity is unknown. The crystal structures of MutT in the apo and holo forms and in the binary and ternary forms complexed with the product 8-oxo-dGMP and 8-oxo-dGMP plus Mn²⁺, respectively, were determined. MutT strictly recognizes the overall conformation of 8-oxo-dGMP through a number of hydrogen bonds. This recognition mode revealed that 8-oxoguanine nucleotides are discriminated from guanine nucleotides by not only the hydrogen bond between the N7-H and Oδ (N119) atoms but also by the syn glycosidic conformation that 8-oxoguanine nucleotides prefer. Nevertheless, these discrimination factors cannot by themselves explain the roughly 34,000-fold difference between the affinity of MutT for 8-oxo-dGMP and dGMP. When the binary complex of MutT with 8-oxo-dGMP is compared with the ligand-free form, ordering and considerable movement of the flexible loops surrounding 8-oxo-dGMP in the binary complex are observed. These results indicate that MutT specifically recognizes 8-oxoguanine nucleotides by the ligand-induced conformational change.     

A nuclear magnetic resonance study of nicotinamide adenine dinucleotide phosphate binding to Lactobacillus casei dihydrofolate reductase.

The binding of NADP+ to dihydrofolate reductase (EC 1.5.1.3) in the presence and absence of substrate analogs has been studied using 1H and 13C nuclear magnetic resonance (NMR). NADP+ binds strongly to the enzyme alone and in the presence of folate, aminopterin, and methotrexate with a stoichiometry of 1 mol of NADP+/mol of enzyme. In the 13C spectra of the binary and ternary complexes, separate signals were observed for the carboxamide carbon of free and bound [13CO]NADP+ (enriched 90% in 13C). The 13C signal of the NADP+-reductase complex is much broader than that in the ternary complex with methotrexate because of exchange line broadening on the binary complex signal. From the difference in line widths (17.5 +/- 3.0 Hz) an estimate of the dissociation rate constant of the binary complex has been obtained (55 +/- 10 sec-1). The dissociation rate of the NADP+-reductase complex is not the rate-limiting step in the overall reaction. In the various complexes studied large 13C chemical shifts were measured for bound [13CO]NADP+ relative to free NADP+ (upfield shifts of 1.6-4.3 ppm). The most likely origin of the bound shifts lies in the effects on the shieldings of electric fields from nearby charged groups. For the NADP+-reductase-folate system two 13C signals from bound NADP+ are observed indicating the presence of more than one form of the ternary complex. The IH spectra of the binary and ternary complexes confirm both the stoichiometry and the value of the dissociation rate constant obtained from the 13C experiments. Substantial changes in the IH spectrum of the protein were observed in the different complexes and these are distinct from those seen in the presence of NADPH.     

Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.     

Uncoupling Kapbeta2 substrate dissociation and ran binding

Karyopherinbeta2 (Kapbeta2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapbeta2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapbeta2-RanGppNHp complex shows that Ran binds in the Kapbeta2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapbeta2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapbeta2 proteolytically (cl-Kapbeta2) and also constructed a mutant of Kapbeta2 with a truncated loop (TL-Kapbeta2). Both modified Kapbeta2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapbeta2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapbeta2 proteins for Ran and substrate. cl-Kapbeta2, TL-Kapbeta2, and native Kapbeta2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapbeta2-Ran or Kapbeta2-substrate interactions. The TL-Kapbeta2 mutant also binds RanGppNHp and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapbeta2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapbeta2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapbeta2-substrate dissociation mechanism where the acidic loop of Kapbeta2 physically couples distinct Ran and substrate binding sites.     

Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome<Emphasis Type="Italic"> c</Emphasis>551i as detected by EPR spectroscopy

EPR studies of the methylamine dehydrogenase (MADH)–amicyanin and MADH–amicyanin–cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry. The binary complex shows a blue copper axial signal, characteristic of oxidized amicyanin. After reaction of substrate with the MADH coenzyme tryptophan tryptophylquinone (TTQ), the binary complex exhibits an equilibrium mixture of oxidized copper/reduced TTQ and reduced copper/TTQ· radical, whose ratio is dependent on the pH. In the oxidized ternary complex, the same copper axial signal is observed superimposed on the low-spin ferric heme features characteristic of oxidized cytochrome c551i. After addition of substrate to the ternary complex, a decrease of the copper signal is observed, concomitant with the appearance of the radical signal derived from the semiquinone form of TTQ. The equilibrium distribution of electrons between TTQ and copper as a function of pH is similar to that observed for the binary complex. This result was essential to establish that the copper center retains its function within the crystalline ternary complex. At high pH, with time the low-spin heme EPR features disappear and the spectrum indicates that full reduction of the complex by substrate has occurred.     

Kinetic mechanism of the EcoRI DNA methyltransferase

We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase). The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with kcat/Km values of 0.51 X 10(8) and 4.1 X 10(8) s-1 M-1, respectively. The Mtase is thus one of the most efficient biocatalysts known. Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition. One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet. Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy). We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound. Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence. Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover.     

Structure of human SMYD2 protein reveals the basis of p53 tumor suppressor methylation

SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel β7 and β8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.     

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid–base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR–Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.     

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.     

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a ternary complex with enzyme/NAD+ similar to enzyme/NAD+/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.     

The mechanism of activation of lipoprotein lipase by apolipoprotein C-II. The formation of a protein-protein complex in free solution and at a triacylglycerol/water interface

The binding of lipoprotein lipase to a fluorescently labelled apolipoprotein C-II in free solution has been followed by measuring fluorescence anisotropy. The formation of a weak, binary complex in which a single apolipoprotein C-II molecule associates non-cooperatively with each subunit of the dimeric enzyme was observed. The dissociation constant for this complex in 0.05 M NaCl is 0.2 X 10(-6) M and it is weakened markedly by raising the salt concentration and by the binding of heparin to the enzyme. The assembly of the same protein-protein complex on the surface of glycerol trioleate globules has been monitored by steady-state and pre-steady-state kinetics. In these circumstances the lipoprotein lipase-apolipoprotein C-II interaction is much tighter (Kd = (7-10) X 10(-9) M) and is insensitive to salt and heparin. The mechanism of activation of the enzyme at low concentrations of apolipoprotein C-II is described by a kinetic model in which apolipoprotein C-II binds preferentially to the form of the enzyme which is associated with the triacylglycerol substrate. This preference leads to a stabilization of the enzyme-substrate complex, thus reducing the apparent Ks.     

5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase

The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods. These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics. Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex. However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate. The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase. The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism.