NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation.

Amino acids in the serine proteinase inhibitor eglin c important for its inhibitory specificity and activity have been investigated by site-directed mutagenesis. The specificity of eglin c could be changed from elastase to trypsin inhibition by the point mutation Leu45----Arg (L45R) in position P1 [nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Model building studies based on the crystal structure of mutant L45R [Heinz et al. (1991) J. Mol. Biol. 217, 353-371] were used to rationalize this specificity change. Surprisingly, the double mutant L45R/D46S was found to be a substrate of trypsin and various other serine proteinases. Multidimensional NMR studies show that wild-type eglin c and the double mutant have virtually identical conformations. In the double mutant L45R/D46S, however, the N-H bond vector of the scissile peptide bond shows a much higher mobility, indicating that the internal rigidity of the binding loop is significantly weakened due to the loss or destabilization of the internal hydrogen bond of the P1' residue. Mutant T44P was constructed to examine the role of a proline in position P2, which is frequently found in serine proteinase inhibitors [Laskowski and Kato (1980) Annu. Rev. Biochem. 49, 593-626]. The mutant remains a potent elastase inhibitor but no longer inhibits subtilisin, which could be explained by model building. Both Arg51 and Arg53, located in the core of the molecule and participating in the hydrogen bonding network with residues in the binding loop to maintain rigidity around the scissile bond, were individually replaced with the shorter but equally charged amino acid lysine. Both mutants showed a decrease in their inhibitory potential. The crystal structure of mutant R53K revealed the loss of two hydrogen bonds between the core and the binding loop of the inhibitor, which are partially restored by a solvent molecule, leading to a decrease in inhibition of elastase by 2 orders of magnitude.     

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.     

Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA.

We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11----Val and Asn15----Val mutations completely abolished, and the Tyr13----Phe and Asn16----Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52----Gln abolished, but Arg52----Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop II and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A----G and G----A replacements in loop II, but not by C----U or U----C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.     

Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I

Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin-like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13-amino acid extension appended to the N terminus. We have determined the solution structure of (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using (15)N nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE). There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average ((1)H)(15)N NOE of 0.55 for the regions. The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C-domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus. Despite these data indicating conformational flexibility for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins.     

Structure of the hirugen and hirulog 1 complexes of alpha-thrombin

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.     

Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg(336) site were replaced with those surrounding Arg(562), were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 9-16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.     

Mutational, NMR, and NH exchange studies of the tight and selective binding of 8-oxo-dGMP by the MutT pyrophosphohydrolase

The solution structure of the ternary MutT enzyme-Mg(2+)-8-oxo-dGMP complex showed the proximity of Asn119 and Arg78 and the modified purine ring of 8-oxo-dGMP, suggesting specific roles for these residues in the tight and selective binding of this nucleotide product [Massiah, M. A., Saraswat, V., Azurmendi, H. F., and Mildvan, A. S. (2003) Biochemistry 42, 10140-10154]. These roles are here tested by mutagenesis. The N119A, N119D, R78K, and R78A single mutations and the R78K/N119A double mutant showed very small effects on k(cat) (相似文献   

Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.     

Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH

Nitric-oxide synthase (NOS) requires the cofactor, (6R)-5,6,7, 8-tetrahydrobiopterin (H4B), for catalytic activity. The crystal structures of NOSs indicate that H4B is surrounded by aromatic residues. We have mutated the conserved aromatic acids, Trp(676), Trp(678), Phe(691), His(692), and Tyr(706), together with the neighboring Arg(414) residue within the H4B binding region of full-length neuronal NOS. The W676L, W678L, and F691L mutants had no NO formation activity and had very low heme reduction rates (<0.02 min(-1)) with NADPH. Thus, it appears that Trp(676), Trp(678), and Phe(691) are important to retain the appropriate active site conformation for H4B/l-Arg binding and/or electron transfer to the heme from NADPH. The mutation of Tyr(706) to Leu and Phe decreased the activity down to 13 and 29%, respectively, of that of the wild type together with a dramatically increased EC(50) value for H4B (30-40-fold of wild type). The Tyr(706) phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Arg(414) to Leu and Glu resulted in the total loss of NO formation activity and of the heme reduction with NADPH. Thus, hydrogen bond networks consisting of the heme carboxylate, Tyr(706), and Arg(414) are crucial in stabilizing the appropriate conformation(s) of the heme active site for H4B/l-Arg binding and/or efficient electron transfer to occur.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

Cellular processing of the interleukin-2 fusion toxin DAB486-IL-2 and efficient delivery of diphtheria fragment A to the cytosol of target cells requires Arg194

We have used site-directed mutagenesis to examine the role played by Arg191, Arg193, and Arg194 of the fusion toxin DAB486-IL-2 in the intoxication of high affinity interleukin-2 receptor-bearing T-lymphocytes. These arginine residues are positioned in the proteolytically sensitive 14-amino acid loop subtended by the disulfide bond between Cys187 and Cys202 in this fusion toxin. DAB486-IL-2 was formed by the genetic substitution of the native diphtheria toxin receptor binding domain with human interleukin-2 (Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B., and Murphy, J.R. (1987) Protein Eng. 1, 493-498). We demonstrate that substitution of Arg194 with Gly results in a 1000-fold loss of DAB486-IL-2 potency. Since trypsin "nicking" of the Gly194 mutant restores biologic activity, we conclude that Arg194 is required for the cellular processing of the fusion toxin which results in the release of fragment A into the cytosol.     

Sheared Aanti.Aanti base pairs in a destabilizing 2 x 2 internal loop: the NMR structure of 5'(rGGCAAGCCU)2

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.     

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu.

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.     

The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase

The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 A. Arginine and asparagine could provide only one hydrogen bond of 2. 52 and 3.02 A, respectively. To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln188-GALT displaced 80 +/- 7. 0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 +/- 8.0 and 129 +/- 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln188-GALT produced 80,030 +/- 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 +/- 71. 2 and 2960 +/- 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal.     

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2)

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.     

Understanding how the distal environment directs reactivity in chlorite dismutase: spectroscopy and reactivity of Arg183 mutants

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.     

Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion. Conversion of ovalbumin to a maspin-like molecule

Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.     

Structural consequences of b- to c-type heme conversion in oxidized Escherichia coli cytochrome b562

An NMR characterization of the 98Arg --> Cys variant of iron (III)-containing cytochrome b562 from Escherichia coli has been performed and the solution structure obtained. This variant has a covalent bond between the heme and Cys 98, thus mimicking the heme binding in cytochrome c. The R98C cytochrome is shown to have a significantly increased stability, compared to that of wild type, toward thermal and chemical denaturation. In water at 20 degrees C it is 5.60 kJ mol-1 more stable than the WT protein, measured by equilibrium guanidine hydrochloride denaturation. The structure has been obtained through two-dimensional total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments and through three-dimensional NOESY-15N heteronuclear multiple quantum coherence (HMQC). By these methods, 85% of protons and 100% of backbone nitrogens were assigned. 2145 meaningful nuclear Overhauser effects (NOEs) (20 NOEs per residue), 45 backbone 3J values, and 397 pseudocontact shifts were used to obtain a family of 35 members, which were then energy-minimized. The root-mean-square deviation (RMSD) with respect to the average structure is 0.50 +/- 0.07 for the backbone and 1.01 +/- 0.08 for the heavy atoms. The magnetic anisotropy resulting from analysis of the pseudocontact shifts indicates an anisotropy that is an intermediate between that of the wild-type, which is the smallest, and cytochrome c. The g values confirm a higher anisotropy of the variant with respect to the wild-type protein. The chirality of the heme 2 alpha carbon is the same as that in all naturally occurring cytochromes c. The overall secondary structure and tertiary structure are very similar to the wild type. The removal of Arg 98 causes a change in the pH-dependent properties. The pKa, proposed to be due to deprotonation of the coordinated histidine, is 1.5 units higher than in the wild type, consistent with the lack of the positive charge of Arg 98 close to the ionizable group. This is further support for the coordinated histidine being the titratable group with an alkaline pKa in the wild-type protein. The pattern of the shifts of the heme methyl groups is different than in the wild-type protein, presumably due to alteration of the electronic structure by the presence of the covalent bond between the protein and the heme. The difference in stability between the variant and wild-type protein is discussed in terms of the structural information.