Glycine-rich Region Research Articles

Site-directed Mutagenesis of Diphosphoinositol Polyphosphate Phosphohydrolase, a Dual Specificity NUDT Enzyme That Attacks Diadenosine Polyphosphates and Diphosphoinositol Polyphosphates

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects.

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within 1 h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Fibrillin assembly: dimer formation mediated by amino-terminal sequences.

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9-11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.

JAZ Requires the Double-stranded RNA-binding Zinc Finger Motifs for Nuclear Localization

We have cloned and characterized a novel zinc finger protein, termed JAZ. JAZ contains four C(2)H(2)-type zinc finger motifs that are connected by long (28-38) amino acid linker sequences. JAZ is expressed in all tissues tested and localizes in the nucleus, primarily the nucleolus. JAZ preferentially binds to double-stranded (ds) RNA or RNA/DNA hybrids rather than DNA. Mutation of individual zinc finger motifs reveals that the zinc finger domains are not only essential for dsRNA binding but are also required for its nucleolar localization, which demonstrates a complex trafficking mechanism dependent on the nucleic acid-binding capability of the protein. Furthermore, forced expression of JAZ potently induces apoptosis in murine fibroblast cells. Thus, JAZ may belong to a class of zinc finger proteins that features dsRNA binding and may regulate cell growth via the unique dsRNA binding properties.

Fibrillin degradation by matrix metalloproteinases: identification of amino- and carboxy-terminal cleavage sites

Fibrillin molecules form the structural framework of elastic fibrillin-rich microfibrils of the extracellular matrix. We have investigated the proteolysis of recombinant fibrillin molecules by five matrix metalloproteinases. Cleavage sites were defined at the carboxy-terminal end of the fibrillin-1 proline-rich region and the corresponding fibrillin-2 glycine-rich region (exon 10), and within exon 49 towards the carboxy-terminus of fibrillin-1. Cleavage at these sites is predicted to disrupt the structure and function of the fibrillin-rich microfibrils.

Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Neighboring glycine residues are essential for P2X 2 receptor/channel function

The roles of a glycine-rich region in the cloned P2X 2 receptor/channel were evaluated by site-directed mutagenesis. Responsiveness to ATP was lost when Gly 247 was replaced by alanine. The sensitivity to ATP was reduced when Gly 248 was replaced by alanine, and the responsiveness to ATP was lost when Gly 248 was replaced by valine. The results suggest that the neighboring glycine residues are essential for P2X 2 receptor/channel function.

Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.

Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.

Cloning of a New Gene (FB19) within HLA Class I Region

A novel gene (named FB19) has been identified within the HLA class I region at human chromosome 6p21.3. A 4.5-kb cDNA containing a 2820-bp open reading frame for a predicted protein of 940 aa was identified. No homology with known gene was detected at the DNA level, while the predicted protein is characterized by a glycine-rich region followed by a domain of 35 residues that shows high homology with the CAT56 gene, another gene of MHC class I. A 4.5-kb transcript was detected in several tissues and cell lines, clearly indicating a wide distribution of expression. Once its function is defined, it could be possible to investigate the relationship between the FB19 gene and the several diseases already mapped within the HLA class I region.

TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.

The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.

Expression of a cysteine-rich protein (CRP) encoding gene during early development of the trout

Members of the cysteine-rich protein (CRP) define a subclass of LIM-only proteins implicated mainly in muscle differentiation. Until now, very little is known concerning the expression of CRP encoding genes during vertebrate development. We describe here the isolation of a trout ( Oncorhynchus mykiss) gene encoding a cysteine-rich protein (TCRP) and the pattern of its mRNA accumulation during embryogenesis, focusing on somitogenesis. TCRP encodes a putative protein with two LIM domains linked to a short glycine-rich region that displays 86%, 76%, 67% identity with chicken CRP2, CRP1 and MLP/CRP3 proteins, respectively. Whole-mount in situ hybridisation showed that TCRP transcript is first detected just before somitogenesis in the paraxial mesoderm, while it is absent in the axial structures. During somitogenesis, the expression of TCRP was observed caudally in the elongating presomitic mesoderm and in the last formed somites. The labelling for TCRP was found to fade as the somites mature. At the end of the somitogenesis, TCRP transcripts accumulation was restricted to pronephros and branchial arches.

The 21-kDa polypeptide (VAP21) in the rabies virion is a CD99-related host cell protein.

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.

Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks

Orb-web weaving spiders rely on their aerial nets to entrap flying prey. A key mechanical feature of orb-web design is the high elasticity of the capture spiral. We report the cloning of substantial cDNA for flagelliform gland silk protein, which forms the core fiber of the catching spiral. Like all silks, the flagelliform protein is composed largely of iterated sequences. The dominant repeat of this protein is Gly-Pro-Gly-Gly-X, which can appear up to 63 times in tandem arrays. This motif likely forms Pro2-Gly3 type II β-turns and the resulting series of concatenated β-turns are thought to form a β-spiral. We propose that this spring-like helix is the basis for the elasticity of silk. The variable fifth position of the motif (X) is occupied by a small subset of residues (Ala, Ser, Tyr, Val). Moreover, these X amino acids occur in specific patterns throughout the repeats. This ordered variation strongly suggests that with hydration, the β-spirals form hydrogen-bonded networks that increase the elasticity of flagelliform silk. The self-assembly of flagelliform protein monomers into silk fibers may be promoted by β-spiral/β-spiral interactions. Additionally, the other two motifs in the flagelliform protein, Gly-Gly-X and a spacer that disrupts the glycine-rich regions, may contribute to the alignment of monomers into fibers.The flagelliform protein cDNA was compared to the other members of the spider silk gene family. We show that all spider silk proteins can be characterized as sets of shared structural modules. The occurrence of these modules among the proteins is inconsistent with the phylogenetic relationships inferred from the C-terminal regions. This observation, along with the high level of variation among individual flagelliform protein repeats, but striking lack of such variation in the other silk proteins, suggests that unusual homogenization processes are involved in silk protein evolution.

A novel splice site mutation of the EXT2 gene in a Finnish hereditary multiple exostoses family. Mutations in brief no. 197. Online.

Hereditary multiple exostoses is a dominantly inherited disease characterized by multiple benign osteochondromas. The affected individuals have an increased risk of developing sarcoma. A large Finnish family with hereditary multiple exostosis was analyzed to find the disease-causing mutation. Blood samples were obtained from 35 family members, including 21 affected and 14 unaffected individuals. Using 2-point linkage analysis the EXT phenotype was shown to be linked to the recently cloned EXT2 gene on chromosome 11p11. The coding region of the gene was sequenced and a previously unreported splice site mutation found. This G to T transversion within a 5-prime splice donor site following exon 6 was shown to cause aberrant splicing of RNA. The described change is considered to be a novel disease-causing mutation in the EXT2 gene.

Ten novel mutations found in Aniridia.

Aniridia (AN) is a sight-threatening congenital ocular disorder characterized by iris hypoplasia, corneal pannus, foveal and optic nerve hypoplasia, cataract formation, and glaucoma. In two-thirds of the patients, AN is inherited in an autosomal dominant fashion with almost complete penetrance but variable expression. The remaining cases are sporadic. Aniridia has been shown to be associated with mutations in the PAX6 gene, located on chromosome 11p13, telomeric to the Wilms' tumor predisposition gene (WT1). This paper describes 14 mutations in the PAX6 gene in patients with AN. Among these 14 mutations, 10 have been unpublished until now. They result most probably in haploinsufficiency and consequently in a reduced protein level of functional PAX6 protein. The mutations reported here are scattered all over the gene, including the paired-box, the glycine-rich region, the homeobox, and the proline-serine-threonine (PST)-rich region.

Plant O-methyltransferases: molecular analysis, common signature and classification.

Comparative analysis of the predicted amino acid sequences of a number of plant O-methyltransferase cDNA clones show that they share some 32-71% sequence identity, and can be grouped according to the different compounds they utilise as substrates. Five highly conserved regions are proposed as a signature for plant O-methyltransferases, two of which (regions I and IV) are believed to be involved in S-adenosyl-L-methionine and metal binding, respectively. The glycine-rich signature regions include a 36 amino acid domain which is located in the mid-terminal section of the carboxy terminus of most O-methyltransferase sequences. Cladistic analysis of the amino acid sequences suggests that plant O-methyltransferases may have arisen from common ancestral genes that were driven by different structural and/or functional requirements, and whose descendants segregated into different biochemical species. A comprehensive classification of plant O-methyltransferases is proposed following the guidelines of the Commission of Plant Gene Nomenclature.

NF-κB p105 Processing via the Ubiquitin-Proteasome Pathway

The p50 subunit of NF-kappa B is generated by proteolytic processing of a 105-kDa precursor (p105) in yeast and mammalian cells. Here we show that yeast mutants in the ubiquitin-proteasome pathway inhibit or abolish p105 processing. Specifically, p105 processing is inhibited by a mutation in a 20 S proteasome subunit (pre1-1), by mutations in the ATPases located in the 19 S regulatory complexes of the proteasome (yta1, yta2/sug1, yta5, cim5), and by a mutation in a proteasome-associated isopeptidase (doa4). A ubiquitinated intermediate of the p105 processing reaction accumulates in some of these mutants, strongly suggesting that ubiquitination is required for processing. However, none of the ubiquitin conjugating enzyme mutants tested (ubc1, -2, -3, -4/5, -6/7, -8, -9, -10, -11) had an effect on p105 processing, suggesting that more than one of these enzymes is sufficient for p105 processing. Interestingly, a mutant "N-end rule" ligase does not adversely affect p105 processing, showing that the N-end rule pathway is not involved in degrading the C-terminal region of p105. Unexpectedly, we found that a glycine-rich region of p105 that is required for p105 processing in mammalian cells is not required for processing in yeast. Thus, p105 processing in both yeast and mammalian cells requires the ubiquitin-proteasome pathway, but the mechanisms of processing, while similar, are not identical.

Tough Tendons: MUSSEL BYSSUS HAS COLLAGEN WITH SILK-LIKE DOMAINS

The primary structure of the alpha-chain of preCol-D (molecular mass = 80 kDa), a tanned collagenous protein predominating in the distal portion of the byssal threads of the mussel Mytilus edulis, was deduced from cDNA to encode an unprecedented natural block copolymer with three major domain types: a central collagen domain flanked by fibroin-like domains and followed by histidine-rich termini. The fibroin-like domains have sequence motifs that strongly resemble the crystalline polyalanine-rich and amorphous glycine-rich regions of spider dragline silk fibroins. The terminal regions resemble the histidine-rich domains of a variety of metal-binding proteins. The silk domains may toughen the collagen by increasing its strength and extensibility. PreCol-D expression is limited to the mussel foot, which contains a longitudinal gradient of preCol-D mRNA. This gradient increases linearly in the proximal to distal direction and reaches a maximum just before the distal depression of the foot.

Analysis of the Quaternary Structure, Substrate Specificity, and Catalytic Mechanism of Valine Dehydrogenase

The solution of the three-dimensional structure of Bacillus sphaericus leucine dehydrogenase has enabled us to undertake a homology-based modeling exercise on the sequence differences between the families of leucine (LeuDH) and valine (ValDH) dehydrogenases. This analysis indicates that the secondary structure elements in the core of the two domains of a single subunit of these enzymes are conserved, as are residues directly implicated in the recognition of the nucleotide cofactor and in catalysis. Comparison of the sequences indicates that the residues in the pocket accommodating the side chain of the amino acid substrate are conserved between these two enzymes, suggesting that the small differences in specificity arise from minor changes in molecular structure, possibly associated with shifts of the main chain rather than mutation of residues in the pocket itself. While B. sphaericus LeuDH is an octamer, both Streptomyces cinnamonensis and Streptomyces coelicolor ValDHs are dimers. The differences in quaternary structure can be understood in terms of the deletion in the latter of a C-terminal loop, which forms important interactions around the four-fold axis in LeuDH.

Identification of an Active Site Alanine in Mevalonate Kinase through Characterization of a Novel Mutation in Mevalonate Kinase Deficiency

Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.