Kinetic and spectroscopic investigations of wild-type and mutant forms of apple 1-aminocyclopropane-1-carboxylate synthase

Two catalytically inactive mutant forms of 1-aminocyclopropane-1-carboxylate (ACC) synthase, Y85A and K273A, were mixed in low concentrations of guanidine hydrochloride (GdnHCl). About 15% of the wild-type activity was recovered (theoretical 25% for a binomial distribution), proving that the functional unit of the enzyme is a dimer, or theoretically, a higher order oligomer. The enzyme catalyzes the conversion of S-adenosyl-L-methionine (SAM) to ACC. The value of kcat/KM is 1.2 x 10(6) M-1 s-1 at pH 8.3. Viscosity variation experiments with glycerol and sucrose as viscosogenic reagents showed that this reaction is nearly 100% diffusion controlled. The sensitivity to viscosity for the corresponding reaction of the less reactive Y233F mutant is much reduced, thus the latter reaction serves as a control for that of the wild-type enzyme. The kcat/KM vs pH profile for wild-type enzyme exhibits pKa values of 7.5 and 8.9. The former is assigned to the pKa of the alpha-amino group of SAM, while the latter corresponds to the independently determined spectrophotometric pKa of the internal aldimine. The kcat vs pH profile exhibits similar pKas, which means that the above pKa values are not perturbed in the Michaelis complex. The phenolic hydroxyl group of Tyr233 forms a hydrogen bond to the 3'-O- of PLP. The spectral and kinetic pKa (kcat/KM) values of the Y233F mutant are not identical (spectral 10.2, kinetic 8.7). A model that accounts quantitatively for these data posits two parallel pathways to the external aldimine for this mutant, the minor one has the alpha-amino group free base form of SAM reacting with the protonated imine form of the enzyme with kcat/KM approximately 6.0 x 10(3) M-1 s-1, while the major pathway involves reaction of the aldehyde form of PLP with SAM with kcat/KM approximately 7.0 x 10(5) M-1 s-1. The spectral pKa is defined only by the less reactive species.     

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.     

Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase

Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. the diiron center is contained in the tmoA polypeptide of teh hydroxylase component [alphabetagamma)2,Mr approximately 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1. Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB. These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.     

Hypertension

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

Genetic complexity, structure, and characterization of highly active bovine intestinal alkaline phosphatases

Mammalian alkaline phosphatases (APs) display 10-100-fold higher kcat values than do bacterial APs. To begin uncovering the critical residues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, approximately 3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, approximately 8, 000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH2-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wild-type and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed average kcat values of 1800, 5900, 4200, and 6100 s-1, respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in kcat. Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its kcat to 1300 s-1, while the converse mutation, i.e. D322G, in bIAP I increased its kcat to 5800 s-1. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex.     

The structure of the monomeric porcine odorant binding protein sheds light on the domain swapping mechanism

The X-ray structure of the porcine odorant binding protein (OBPp) was determined at 2.25 A resolution. This lipocalin is a monomer and is devoid of naturally occurring bound ligand, contrary to what was observed in the case of bovine OBP [Tegoni, M., et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, M. A., et al. (1996) Nat. Struct. Biol. 3, 934-939]. In this latter protein, a dimer without any disulfide bridges, domain swapping was found to occur between the beta- and alpha-domains. A single Gly (121) insertion was found in OBPp when it was compared to OBPb, which may prevent domain swapping from taking place. The presence of a disulfide bridge between the OBPp beta- and alpha-domains (cysteines 63 and 155) may lock the resulting fold in a nonswapped monomeric conformation. Comparisons with other OBPs indicate that the two cysteines involved in the OBPp disulfide bridge are conserved in the sequence, suggesting that OBPp may be considered a prototypic OBP fold, and not OBPb.     

Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a Kcat of 14.3 s-1 and a kcat/KM of 2.70 x 10(6) s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.     

Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity

The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.     

Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.     

Mechanisms of inhibition of amido phosphoribosyltransferase from mouse L1210 leukemia cells

Amido phosphoribosyltransferase (amido PRTase) catalyses the first step of the pathway for de novo biosynthesis of purine nucleotides. The enzyme is subject to inhibition by purine nucleoside 5'-monophosphates (AMP, IMP, and GMP), by dihydrofolate polyglutamates, and by the antifolate piritrexim [Sant, M. E., Lyons, S. D., Phillips, L., & Christopherson, R. I. (1992) J. Biol. Chem. 267, 11038-11045). Using a coupled radioassay, we have determined the substrate dissociation constants as 80.4 +/- 13.2 microM for 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) and 421 +/- 193 microM for L-glutamine with P-Rib-PP bound first with positive cooperativity for interaction with a second site on the catalytically active dimer (interaction factor of 0.247 +/- 0.042). Analysis of inhibition patterns for amido PRTase shows that the antifolate piritrexim is a noncompetitive inhibitor bound with positive cooperativity at two allosteric sites of an inactive dimer with a dissociation constant of 66.0 +/- 17.8 microM for interaction with the free enzyme and an interaction factor of 0.187 +/- 0.113 with P-Rib-PP as the varied substrate. With L-glutamine as the varied substrate, a dissociation constant of 62.3 +/- 15.6 microM for interaction with the enzyme-P-Rib-PP complex and an interaction factor of 0.0958 +/- 0.0585 microM were obtained. AMP binds as a competitive inhibitor with respect to P-Rib-PP with a dissociation constant of 40.0 +/- 8.1 microM for interaction with the free enzyme and as a noncompetitive inhibitor with respect to L-glutamine with a dissociation constant of 16.4 +/- 5.2 mM for interaction with the enzyme-P-Rib-PP complex. Sucrose density gradient centrifugation of partially purified amido PRTase showed three molecular forms of the enzyme: an inactive tetramer (10.2 S) formed in the presence of AMP, an active dimer (6.7 S) formed with P-Rib-PP, and an inactive dimer (7.2 S) with piritrexim. The latter species may predominate in cells containing high levels of dihydrofolate polyglutamates.     

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.     

Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors

Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).     

Pro region C-terminus:protease active site interactions are critical in catalyzing the folding of alpha-lytic protease

alpha-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L. , and Agard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The pro region also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the active site by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904]. Through mutagenesis and structure-function analyses we have begun to investigate the mechanism by which the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Of central interest has been mapping the interface between the pro region and the protease and identifying interactions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletions of the pro region were found to have drastic effects on the rate at which the pro region folds the protease but surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly support a model in which the detailed interactions between the pro region C-terminus and the protease are remarkably similar to those of known substrate/inhibitor complexes. Further, mutation of two protease residues near the active site have significant effects on stabilization of the folding transition state (kcat) or in binding to the folding intermediate (KM). Our results suggest a model for the alpha-lytic protease pro region-mediated folding reaction that may be generally applicable to other pro region-dependent folding reactions.     

Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.     

Domain swapping in G-protein coupled receptor dimers

Computer simulations were performed on models of the beta2-adrenergic receptor dimer, including 5,6-domain swapped dimers which have been proposed as the active, high affinity form (here the dimer interface lies between helices 5 and 6). The calculations suggest that the domain swapped dimer is a high energy structure in both the apo dimer and in the presence of propranolol. In the presence of agonist the energy of the domain swapped dimer is significantly lowered. Analysis of the dimer structure suggests that the agonist-induced conformational change optimizes the helix-helix interactions at the 5-6 interface. An antagonist on the other hand has little effect on these interactions. These observations are consistent with the hypothesis that the agonist functions by shifting the equilibrium in favour of the domain swapped dimer. Indirect support for the domain swapping hypothesis was obtained from the correlated mutations amongst the external residues of the known beta2-adrenergic receptors. These occur mainly at the 5-6 interface at precisely the locations predicted by the simulations; site-directed mutagenesis data in support of a functional role for these lipid-facing correlated residues is presented. The article includes a review of the experimental evidence for G-protein coupled receptor dimerization. Many other aspects of G-protein coupled receptor activation are discussed in terms of this domain swapping hypothesis     

Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I

NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.     

Characterization of recombinant CD45 cytoplasmic domain proteins. Evidence for intramolecular and intermolecular interactions

CD45 is a transmembrane two-domain tyrosine phosphatase required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain phosphatases, the role of the second phosphatase domain is unclear. In this study, recombinant CD45 cytoplasmic domain proteins purified from bacteria were used to evaluate the function of the individual phosphatase domains. A recombinant protein expressing the membrane-proximal region, first phosphatase domain, and spacer region of CD45 (rD1) was catalytically active and found to exist primarily as a dimer. In contrast to this, a recombinant protein expressing the spacer region, the second phosphatase domain and the carboxy tail of CD45 (rD2) existed as a monomer and had no catalytic activity against any of the substrates tested. Comparison of rD1 with the recombinant protein expressing the entire cytoplasmic domain of CD45 (rD1/D2) indicated that rD1/D2 was 2-3-fold more catalytically active, was more thermostable, and existed primarily as a monomer. Limited trypsin digestion of rD1/D2 provided evidence for a noncovalent association between an N-terminal 27-kDa fragment and a C-terminal 53-kDa fragment, suggesting an intramolecular interaction. Furthermore, rD1 was found to specifically associate with rD2 in an in vitro binding assay. Taken together, these data provide evidence for an intramolecular interaction occurring in the cytoplasmic domain of CD45. In the absence of the C-terminal region containing the second phosphatase domain, intermolecular interactions occur, resulting in dimer formation.     

Reaction mechanism of Amphibacillus xylanus NADH oxidase/alkyl hydroperoxide reductase flavoprotein

NADH oxidase from Amphibacillus xylanus is a potent alkyl hydroperoxide reductase in the presence of the small disulfide-containing protein (AhpC) of Salmonella typhimurium. In the presence of saturating AhpC, kcat values for reduction of hydroperoxides are approximately 180 s-1, and the double mutant flavoprotein enzyme C337S/C340S cannot support hydroperoxide reduction (Niimura, Y., Poole, L. B., and Massey, V. (1995) J. Biol. Chem. 270, 25645-25650). Kinetics of reduction of wild-type and mutant enzymes are reported here with wild-type enzyme; reduction by NADH was triphasic, with consumption of 2.6 equivalents of NADH, consistent with the known composition of one FAD and two disulfides per subunit. Rate constants for the first two phases (each approximately 200 s-1) where FAD and one disulfide are reduced are slightly greater than kcat values for AhpC-linked hydroperoxide reduction. The rate constant for the third phase (reduction to the 6-electron level) is too small for catalysis. Only the first phase of the wild-type enzyme occurs with the mutant enzyme. These results and the stoichiometry of NADH consumption indicate Cys337 and Cys340 as the active site disulfide of the flavoprotein and that electrons from FADH2 must pass through this disulfide to reduce the disulfide of AhpC.     

Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy

Two dimensional 1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115-131-amide peptide (TnI115-131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115-131 to N-TnC in low salt and 100 mM KCl samples was determined to be 28 +/- 4 and 24 +/- 4 microM, respectively. The off rate of TnI115-131 was determined to be 300 s-1 from observed N-TnC backbone amide 1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330-8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115-131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the "hydrophobic pocket" (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784-789).