Crystal structure of wild-type tryptophan synthase complexed with the natural substrate indole-3-glycerol phosphate

We used freeze trapping to stabilize the Michaelis complex of wild-type tryptophan synthase and the alpha-subunit substrate indole-3-glycerol phosphate (IGP) and determined its structure to 1. 8 A resolution. In addition, we determined the 1.4 A resolution structure of the complex with indole-3-propanole phosphate (IPP), a noncleavable IGP analogue. The interaction of the 3'-hydroxyl of IGP with the catalytic alphaGlu49 leads to a twisting of the propane chain and to a repositioning of the indole ring compared to IPP. Concomitantly, the catalytic alphaAsp60 rotates resulting in a translocation of the COMM domain [betaGly102-betaGly189, for definition see Schneider et al. (1998) Biochemistry 37, 5394-5406] in a direction opposite to the one in the IPP complex. This results in loss of the allosteric sodium ion bound at the beta-subunit and an opening of the beta-active site, thereby making the cofactor pyridoxal 5'-phosphate (PLP) accessible to solvent and thus serine binding. These findings form the structural basis for the information transfer from the alpha- to the beta-subunit and may explain the affinity increase of the beta-active site for serine upon IGP binding.     

Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E. coli transhydrogenase

The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266. A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values. It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.     

The three-dimensional structure of Asp189Ser trypsin provides evidence for an inherent structural plasticity of the protease.

Trypsin mutant Asp189Ser, first described by Gráf et al. [Gráf, L., Jancsó, A., Szilágyi, L., Hegyi, G., Pintér, K., Náray-Szabó, G., Hepp, J., Medzihradszky, K. & Rutter, W.J. (1988) Proc. Natl Acad. Sci. USA 85, 4961-4965] has played an important role in recent studies on the structural basis of substrate-specific catalysis by serine proteases. The present work reports the three-dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X-ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X-ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild-type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural-functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substrate-specific serine protease action.     

Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.

Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.     

Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha-linked galactose from a P(k) epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633). Using a synthetic P(k)-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both alpha-2,3 and alpha-2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the alpha-2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the alpha-2,6-sialyltransferase activity in vitro as well as in vivo.     

Multiconformational states in phosphoglycerate dehydrogenase

Phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in the serine biosynthetic pathway. In lower plants and bacteria, the PGDH reaction is regulated by the end-product of the pathway, serine. The regulation occurs through a V(max) mechanism with serine binding and inhibition occurring in a cooperative manner. The three-dimensional structure of the serine inhibited enzyme, determined by previous work, showed a tetrameric enzyme with 222 symmetry and an unusual overall toroidal appearance. To characterize the allosteric, cooperative effects of serine, we identified W139G PGDH as an enzymatically active mutant responsive to serine but not in a cooperative manner. The position of W139 near a subunit interface and the active site cleft suggested that this residue is a key player in relaying allosteric effects. The 2.09 A crystal structure of W139G-PGDH, determined in the absence of serine, revealed major quaternary and tertiary structural changes. Contrary to the wildtype enzyme where residues encompassing residue 139 formed extensive intersubunit contacts, the corresponding residues in the mutant were conformationally flexible. Within each of the three-domain subunits, one domain has rotated approximately 42 degrees relative to the other two. The resulting quaternary structure is now in a novel conformation creating new subunit-to-subunit contacts and illustrates the unusual flexibility in this V(max) regulated enzyme. Although changes at the regulatory domain interface have implications in other enzymes containing a similar regulatory or ACT domain, the serine binding site in W139G PGDH is essentially unchanged from the wildtype enzyme. The structural and previous biochemical characterization of W139G PGDH suggests that the allosteric regulation of PGDH is mediated not only by changes occurring at the ACT domain interface but also by conformational changes at the interface encompassing residue W139.     

Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure

The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.     

Roles of individual amino acids in helix 14 of the membrane domain of proton-translocating transhydrogenase from Escherichia coli as deduced from cysteine mutagenesis

Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane alpha helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues betaLeu240 to betaSer260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of betaGly245, betaGly249, and betaGly252, also to leucines. In addition to the residues forming the helix, residues betaAsn238 and betaAsp239 were also mutated. Except for betaI242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines betaGly245, betaGly249, and betaGly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of betaGly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines betaSer250, betaSer251, and betaSer256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.     

Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. Histidine 142 alpha is a facilitative catalytic residue

There are 11 histidine residues in Escherichia coli succinyl-CoA synthetase. His-246 alpha is well established as the phosphorylation site of the enzyme. Replacement of this histidine by asparagine (Mann, C. J., Mitchell, T., and Nishimura, J. S. (1991) Biochemistry 30, 1497-1503) or by aspartic acid (Majumdar, R., Guest, J. R., and Bridger, W. A. (1991) Biochim. Biophys. Acta 1076, 86-90) through site-directed mutagenesis resulted in complete loss of enzyme activity. Chemical modification experiments suggested a second histidine at the active site (Collier, G. E., and Nishimura, J. S. (1979) J. Biol. Chem. 254, 10925-10930). In the present study, we have changed His-142 alpha to an asparagine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is practically devoid of enzyme activity but can be thiophosphorylated with adenosine 5'-O-(thiotriphosphate) and dethiophosphorylated with ADP at rates that are significantly faster than those with wild type enzyme. The observation that phosphorylated mutant enzyme can be dephosphorylated with succinate and with succinate plus desulfo-CoA at rates comparable with those with wild type enzyme suggests that mutant enzyme can bind succinate and CoA. Dethiophosphorylation of the enzyme in the presence of CoA plus succinate proceeds much faster with wild type than with mutant. While there was no significant change in KCoA or Ksuccinate, the turnover number for dethiophosphorylation of the mutant was 10-fold lower. These data are consistent with location of His-142 alpha at the active site and a facilitative role for this residue in catalysis.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

Phosphorylation of smooth muscle caldesmon by calmodulin-dependent protein kinase II. Identification of the phosphorylation sites

Smooth muscle caldesmon was phosphorylated by smooth muscle calmodulin-dependent protein kinase II. The extent of phosphorylation obtained was 5.65 mol of phosphate/mol of caldesmon. Phosphorylated protein was subjected to the complete trypsin proteolysis and the produced phosphopeptides were purified by C-8 reverse phase chromatography. Nine phosphopeptides were isolated and by amino acid sequence analysis, eight phosphorylation sites were identified. According to the published amino acid sequence of chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879), these sites were serine 26, serine 59, serine 73, threonine 469, serine 475, serine 587, serine 620, and serine 726. The time course of phosphorylation of these sites was also measured and it was concluded that the first site was serine 73, the second site was serine 26, the third site was serine 726, and the fourth site was serine 587. The preferred phosphorylation sites were located in the amino terminus myosin binding domain whereas slower phosphorylation occurred in the carboxyl terminus actin/calmodulin domain.     

Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis

We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.     

Intrinsic fluorescence of succinyl-CoA synthetase and four tryptophan mutants. Tryptophan 76 and tryptophan 248 of the beta-subunit are responsive to CoA binding

Previous studies showed that modification of an average of one of the three tryptophan residues of succinyl-CoA synthetase of Escherichia coli abolished enzyme activity, but did not prevent phosphorylation of the enzyme by ATP [Ybarra, J., Prasad, A. R. S., & Nishimura, J.S. (1986) Biochemistry 25, 7174-7178]. In the present study, single mutations in which each of the three tryptophans (beta-Trp43, beta-Trp76, and beta-Trp248) has been changed to phenylalanine (designated W43F, W76F, and W248F) have been accomplished by the technique of site-directed mutagenesis and the mutant proteins isolated. In addition, a double mutant in which beta-Trp43 and beta-Trp248 were changed to phenylalanines (W43,248F) has also been isolated. Each of the mutant enzymes was practically as active as wild type. Since the emission spectrum of beta-Trp76 reflected a low fluorescence intensity for this residue, it was possible to obtain the emission spectrum of each tryptophan residue by using W43F, W248F, and W43,248F. From the positions of the emission maxima and the results of iodide quenching of fluorescence, it was deduced that beta-Trp248 is a surface residue, beta-Trp43 is buried, and beta-Trp76 is intermediate in location. Coenzyme A, but no other substrate, protected the fluorescence of beta-Trp76 and beta-Trp248, but not of beta-Trp43, against quenching by acrylamide. These results are consistent with an interaction between beta-Trp76 and beta-Trp248 and the binding site for CoA.     

Structure of UDP complex of UDP-galactose:beta-galactoside-alpha -1,3-galactosyltransferase at 1.53-A resolution reveals a conformational change in the catalytically important C terminus

UDP-galactose:beta-galactosyl alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-alpha-d-galactose into an alpha-1,3 linkage with beta-galactosyl groups in glycoconjugates. The enzyme is expressed in many mammalian species but is absent from humans, apes, and old world monkeys as a result of the mutational inactivation of the gene; in humans, a large fraction of natural antibodies are directed against its product, the alpha-galactose epitope. alpha3GT is a member of a family of metal-dependent retaining glycosyltransferases including the histo-blood group A and B synthases. A crystal structure of the catalytic domain of alpha3GT was recently reported (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). However, because of the limited resolution (2.3 A) and high mobility of the atoms (as indicated by high B-factors) this structure (form I) does not provide a clear depiction of the catalytic site of the enzyme. Here we report a new, highly ordered structure for the catalytic domain of alpha3GT at 1.53-A resolution (form II). This provides a more accurate picture of the details of the catalytic site that includes a bound UDP molecule and a Mn(2+) cofactor. Significantly, in the new structure, the C-terminal segment (residues 358-368) adopts a very different, highly structured conformation and appears to form part of the active site. The properties of an Arg-365 to Lys mutant indicate that this region is important for catalysis, possibly reflecting its role in a donor substrate-induced conformational change.     

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe-protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884-1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe-protein docking surface of the Fe-protein. Therefore, it is possible that the loss of function by A. vinelandii UW97-Fe-protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe-protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen-free medium. One such revertant, designated A vinelandii BG9, encoded a Fe-protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe-protein. This region is implicated in stabilizing the MgATP-induced conformation of the Fe-protein and in docking to the MoFe-protein. Further complementation analysis showed that the Fe-protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non-functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe-protein were functional; however, the Fe-protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.     

Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.     

Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.     

Kinetic studies on the interaction of alpha 1-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases

The rates of interaction of a number of serine proteinases with a mutant form of alpha 1-proteinase inhibitor (referred to as alpha 1-proteinase inhibitor (Pittsburgh)), in which a methionine-358 to arginine-358 mutation has occurred, have been determined. An approximately 6,000-fold increase in the second order association rate constant with human thrombin was observed (48 M-1 X s-1 for the normal protein to 3.1 X 10(5) M-1 X s-1 for the arginine mutant), confirming previously observed data using bovine thrombin (Owen, M.C., Brennan, S.O., Lewis, J.H. & Carrell, R.W. (1983) New England J. Med. 309, 694-698). However, substantial increases in the rates of association with other trypsin-like enzymes were also noted, indicating that the replacement of methionine by a basic residue affects all serine proteinases with this kind of specificity. There was a marked decrease in the rates of interaction of the Pittsburgh mutant with both human neutrophil elastase and porcine pancreatic elastase, the inhibitor being converted into lower molecular mass fragments after interaction with either enzyme. Butanedione caused a substantial loss in the inhibitory activity of the arginine mutant, while having no effect on the normal protein. These data, when compared to those previously reported for differences in reaction rates between normal and oxidized alpha 1-proteinase inhibitor (Beatty, K., Bieth, J. & Travis, J. (1980) J. Biol. Chem. 255, 3931-3934), are consistent with the interpretation that the amino acid in the P1-position at the reactive site of this protein has a marked effect on determining its primary specificity.     

Smooth muscle myosin light chain kinase. Amino acid sequence at the site phosphorylated by adenosine cyclic 3',5'-phosphate-dependent protein kinase whether or not calmodulin is bound

Turkey gizzard smooth muscle myosin light chain kinase is a calmodulin-dependent enzyme containing 2 serine residues that can be phosphorylated by cAMP-dependent protein kinase. One of these sites can be phosphorylated only when calmodulin is not bound to the enzyme; the amino acid sequence around this site has been reported recently (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464). Here we report the sequence around the site that is phosphorylated by cAMP-dependent protein kinase whether or not calmodulin is bound: Lys-Ala-Ser(P)-Gly-Ser-Ser-Pro-Thr-Ser-Pro-Ile-Asn-Ala-Asp-Lys-Val-Glu-A sn-Glu- . This sequence conforms to the previously defined criteria for substrates of cAMP-dependent protein kinase.