Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.     

Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity.

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.     

Sequence of rat lipoprotein lipase-encoding cDNA.

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.     

The role of the environment around the catalytic triad in β-trypsin: A molecular orbital study

In the enzymatic reaction of β-trypsin the role of environment around the catalytic triad is studied by means of ab initio molecular orbital calculations. The triple ion form of the catalytic triad (Asp 102(?)-His 57(+)-Ser 195(?)) is considerably more stable than the double proton-transferred form (Asp 102(neutral)-His 57(neutral)-Ser 195(?)), due to the environment around it, rather than its nature. The “electrostatic mechanism” is more favorable than the “charge relay mechanism” owing to the nature of the enzyme as a biopolymer.     

A missense mutation (Trp86----Arg) in exon 3 of the lipoprotein lipase gene: a cause of familial chylomicronemia.

We have investigated a patient of English ancestry with familial chylomicronemia caused by lipoprotein lipase (LPL) deficiency. DNA sequence analysis of all exons and intron-exon boundaries of the LPL gene identified two single-base mutations, a T----C transition for codon 86 (TGG) at nucleotide 511, resulting in a Trp86----Arg substitution, and a C----T transition at nucleotide 571, involving the codon CAG encoding Gln106 and producing Gln106----Stop, a mutation described by Emi et al. The functional significance of the two mutations was confirmed by in vitro expression and enzyme activity assays of the mutant LPL. Linkage analysis established that the patient is a compound heterozygote for the two mutations. The Trp86----Arg mutation in exon 3 is the first natural mutation identified outside exons 4-6, which encompass the catalytic triad residues.     

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase.

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.     

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to delta-opioid receptor independently of receptor binding.

Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity

Photoaffinity labeling experiments with diphtheria toxin fragment A have implicated glutamic acid 148 as a constituent of the NAD binding site. To evaluate the role of this residue in ADP-ribosylation of elongation factor 2, we replaced it with aspartic acid by in vitro mutagenesis of a toxin gene fragment cloned in Escherichia coli. Fragment A containing aspartic acid at position 148 had less than 0.6% the ADP-ribosylation activity of wild-type fragment A. The mutation produced no change in sensitivity of fragment A to trypsin and little, if any, reduction in affinity of fragment A for NAD. These results indicate that glutamic acid 148 is essential for the ADP-ribosylation of elongation factor 2 and are consistent with other data suggesting that this residue may be at or near the catalytic center of the toxin.     

Critical aspartic acid residues in pseudouridine synthases.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.     

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.     

The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.     

A domain of the manganese-stabilizing protein from Synechococcus elongatus involved in functional binding to photosystem II.

Site-directed mutagenesis was performed to investigate whether the two protease-sensitive sequences Phe(156)-Gly(163) and Arg(184)-Ser(191), of the manganese-stabilizing protein (MSP) from a thermophilic cyanobacterium, Synechococcus elongatus (Motoki, A., Shimazu, T., Hirano, M., and Katoh, S. (1998) Biochim. Biophys. Acta 1365, 492-502), are involved in functional interaction with photosystem II (PSII). The ability of MSP to bind to its functional site on the PSII complex and to reactivate oxygen evolution was dramatically reduced by the substitution of Arg(152), Asp(158), Lys(160), or Arg(162) with uncharged residues, by insertion of a single residue between Phe(156) and Leu(157), or by deletion of Leu(157). Substitution of each of the four charged residues with an identically charged residue showed that the charges at Asp(158), and possibly Lys(160), are important for the electrostatic interaction with PSII. The reactivating ability was also strongly affected by the alteration of Phe(156) to Leu. Replacement of Lys(188), the only strictly conserved charged residue in the Arg(184)-Ser(191) sequence, by Gln had only a marginal effect on the function of MSP. High affinity binding of MSP to PSII was also affected significantly by mutation at Arg(152), which is located in a region (Val(148)-Arg(152)) strictly conserved among the 14 sequences so far reported. These results imply that the Val(148)-Gly(163) sequence, which is well conserved among MSPs from cyanobacteria to higher plants, is a domain of MSP for functional interaction with PSII.     

Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.     

Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59.

Threonine 59, a helix-capping residue at the amino terminus of the longest helix in T4 phage lysozyme, was substituted with valine, alanine, glycine, serine, asparagine, and aspartic acid. The valine, alanine, and glycine replacements were observed to be somewhat more destabilizing than serine, asparagine, and aspartic acid. The crystal structures of the different variants showed that changes in conformation occurred at the site of substitution, including Asp 61, which is nearby, as well as displacement of a solvent molecule that is hydrogen-bonded to the gamma-oxygen of Thr 59 in wild-type lysozyme. Neither the structures nor the stabilities of the mutant proteins support the hypothesis of Serrano and Fersht (1989) that glycine and alanine are better helix-capping residues than valine because a smaller-sized residue allows better hydration at the end of the helix. In the aspartic acid and asparagine replacements the substituted side chains form hydrogen bonds with the end of the helix, as does threonine and serine at this position. In contrast, however, the Asp and Asn side chains also make unusually close contacts with carbon atoms in Asp 61. This suggests a structural basis for the heretofore puzzling observations that asparagine is more frequently observed as a helix-capping residue than threonine [Richardson, J. S., & Richardson, D. C. (1988) Science 240, 1648-1652] yet Thr----Asn replacements at N-cap positions in barnase were found to be destabilizing [Serrano, L., & Fersht, A. R. (1989) Nature 342, 296-299].(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamHI sites. This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use in kinetic, crystallographic and NMR experiments. Aspartate 372 is an invariant amino acid residue in genes known to code for a functionally active PGK. The function of this acidic residue appears to be to help desolvate the magnesium ion complexed with either ADP or ATP when this substrate binds to the enzyme. Both crystallographic and nuclear magnetic resonance experiments show that the replacement of the residue with asparagine has only minimal effects on the overall structure. The substitution of the charged carboxyl group with that of the neutral amide affects the binding of the nucleotide substrate as predicted but not, as might have been expected, the binding of 3-phosphoglycerate. The overall velocity of the enzymic reaction (Vmax) is reduced 10-fold by the substitution of aspartic acid 372 by an asparagine residue (D372N). This reduction in Vmax is considerably less than one would expect from its known position within the structure of the enzyme. This result therefore poses questions about our understanding of charged groups at the active centres of enzymes and of the reason for their apparent conservation.