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PubMed2001

Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals.

Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA. We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DNA substrates were prepared by gamma-irradiation of DNA solutions under N(2)O or air, such that they contained a multiplicity of modified bases. Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases. Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics. The kinetic parameters also depended on the identity of the individual modified base being removed. Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90-->Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study. This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates. The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.

DNA Damage↗
PubMed1990

Characterization of two tyrosine-specific protein kinases from pig spleen. Substrate-specific effect of autophosphorylation.

Spontaneously active tyrosine-specific protein kinases I and II (designated TyrK I and TyrK II) have been purified to electrophoretic homogeneity from a particulate fraction of porcine spleen based on an assay that used poly(4Tyr, Glu) as a substrate. SDS/polyacrylamide gels revealed a doublet of bands of about Mr 51,000 for TyrK I and two protein bands of Mr 55,000 and 54,000 for TyrK II. After incubation in the presence of [gamma-32P]ATP, the bands corresponding to both protein kinases contained phosphotyrosine. The two tyrosine protein kinases showed high activities with poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu) as substrates and lower activity with angiotensin II. Neither histone, phosvitin, casein nor bovine serum albumin were phosphorylated. Both protein tyrosine kinases were activated by millimolar concentrations of Mg2+ whereas Mn2+ was less effective. The effects of various polyanionic and polycationic substances depended on the nature of the peptide substrate. With poly(Tyr, 4Glu) as a substrate, the substances either inhibited the activities of TyrK I and TyrK II or had no effect. However, activation was observed with angiotensin II as substrate in the presence of polylysine, polyornithine, protamine sulfate, and heparin as effectors. When angiotensin II was used as substrate, activation also occurred by autophosphorylation, in parallel to the phosphate incorporation into the protein kinases. Activation by autophosphorylation was not observed with the synthetic peptide substrates, poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu).

Animals↗
PubMed2004

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases.

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.

Amino Acid Sequence↗
PubMed2003

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity.

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

Crystallography, X-Ray↗
PubMed1975

4-Nitrobenzoic acid reductase of Ascaris lumbricoides var suum. Substrate specificity and reaction products.

1. The substrate specificity of nitro-reductase from Ascaris lumbricoides varsum was determined. This enzyme reduced nitrobenzene, 4-nitrohippuric acid and the isomers of nitrophenol, nitroanisole, nitrobenzoic acid, nitrobenzaldehyde and nitrobenzyl alcohol. The same enzyme preparation reduced azobenzene, 4-dimethylaminoazobenzene and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene. Nitrobenzaldehyde isomers were not reduced to the alcohols. 2. The products of nitro- and azo-reduction were the corresponding amines, no hydroxylamino or hydrazo compounds were detected. 3. The pH optima and cofactor requirements were the same for both azo- and nitro-reduction and neither reaction was inhibited by oxygen. 4. Ammonium sulphate fractionation failed to separate azo- and nitro-reductase activities. The molecular weight of both azo- and nitro-reductase was about 130 000.

Amines↗
PubMed2002

Factors governing nonoverlapping substrate specificity by mitochondrial inner membrane peptidase.

At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins. One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae. Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities. A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the -1 and -3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides. We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position. We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p. A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases.

Intracellular Membranes↗
PubMed2001

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.

Binding Sites↗
PubMed2004

Defining substrate specificity and catalytic mechanism in ascorbate peroxidase.

Haem peroxidases catalyse the H2O2-dependent oxidation of a variety of, usually organic, substrates. Mechanistically, these enzymes are very well characterized: they share a common catalytic cycle that involves formation of a two-electron oxidized intermediate (Compound I) followed by reduction of Compound I by substrate. The substrate specificity is more diverse, however. Most peroxidases oxidize small organic substrates, but there are prominent exceptions to this and the structural features that control substrate specificity remain poorly defined. APX (ascorbate peroxidase) catalyses the H2O2-dependent oxidation of L-ascorbate and has properties that place it at the interface between the class I (e.g. cytochrome c peroxidase) and classical class III (e.g. horseradish peroxidase) peroxidase enzymes. We present a unified analysis of the catalytic and substrate-binding properties of APX, including the crystal structure of the APX-ascorbate complex. Our results provide new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase-mediated catalysis for more than 20 years.

Ascorbate Peroxidases↗
PubMed2001

Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3.

Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are involved in the absorption and elimination of a wide variety of structurally unrelated amphipathic organic compounds. Several members of this protein family mediate the uptake of substrates across the basolateral membrane of hepatocytes as the first step in hepatic elimination. In contrast to the well-characterized Oatp1 and Oatp2, the localization and substrate specificity of the recently cloned Oatp4 have not been investigated in detail. Therefore, we raised an antibody against the C-terminal end of Oatp4 and localized this 85-kDa protein to the basolateral membrane of rat hepatocytes. Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for bromosulfophthalein, dehydroepiandrosterone sulfate, leukotriene C4, and anionic peptides. In addition, we compared the substrate specificity of Oatp4 to that of Oatp3, which so far has mainly been shown to mediate intestinal bile acid transport. Oatp3 had a similar broad substrate specificity, but in general much lower affinities than Oatp4. Thus, while Oatp4 seems to work in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine.

Animals↗
PubMed2004

In vivo substrate specificity of periplasmic disulfide oxidoreductases.

In Escherichia coli, a family of periplasmic disulfide oxidoreductases catalyzes correct disulfide bond formation in periplasmic and secreted proteins. Despite the importance of native disulfide bonds in the folding and function of many proteins, a systematic investigation of the in vivo substrates of E. coli periplasmic disulfide oxidoreductases, including the well characterized oxidase DsbA, has not yet been performed. We combined a modified osmotic shock periplasmic extract and two-dimensional gel electrophoresis to identify substrates of the periplasmic oxidoreductases DsbA, DsbC, and DsbG. We found 10 cysteine-containing periplasmic proteins that are substrates of the disulfide oxidase DsbA, including PhoA and FlgI, previously established DsbA substrates. This technique did not detect any in vivo substrates of DsbG, but did identify two substrates of DsbC, RNase I and MepA. We confirmed that RNase I is a substrate of DsbC both in vivo and in vitro. This is the first time that DsbC has been shown to affect the in vivo function of a native E. coli protein, and the results strongly suggest that DsbC acts as a disulfide isomerase in vivo. We also demonstrate that DsbC, but not DsbG, is critical for the in vivo activity of RNase I, indicating that DsbC and DsbG do not function identically in vivo. The absence of substrates for DsbG suggests either that the in vivo substrate specificity of DsbG is more limited than that of DsbC or that DsbG is not active under the growth conditions tested. Our work represents one of the first times the in vivo substrate specificity of a folding catalyst system has been systematically investigated. Because our methodology is based on the simple assumption that the absence of a folding catalyst should cause its substrates to be present at decreased steady-state levels, this technique should be useful in analyzing the substrate specificity of any folding catalyst or chaperone for which mutations are available.

Cysteine↗
PubMed1996

Substrate specificity of alpha-glucuronidase isolated from snail acetone powder.

The substrate specificity of a p-nitrophenyl alpha-D-glucopyranosyl-uronic acid-hydrolyzing enzyme (PNP-GAase) isolated from snail acetone powder has been investigated with various substrates, such as p-nitrophenyl alpha-D-glucopyranosyluronic acid (PNP-GA), 2-O-alpha- D-glucopyranosyluronic acid-D-xylose (GA-2X), 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylose (MeGA-2X), and O-alpha-D-glucopyranosyluronic acid-alpha-D-glucopyranosiduronic acid (GA-GA). The Km (mM) and Vmax (mumol of glucuronic acid formed/mg of enzyme protein/min) toward these substrates were as follows; 0.13 and 3.21 for PNP-GA, 0.33 and 0.089 for GA-2X, 17.6 and 0.094 for MeGA-2X, and 0.36 and 0.015 for GA-GA, respectively. The results indicate that the PNP-GAase specifically hydrolyzed PNP-GA, however, the enzyme had broad substrate specificity.

Acetone↗
PubMed1996

Substrate specificity of IphP, a cyanobacterial dual-specificity protein phosphatase with MAP kinase phosphatase activity.

The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.

Adenosine Diphosphate↗
PubMed2003

Analysis of a substrate specificity switch residue of cephalosporin acylase.

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.

Adipates↗
PubMed2006

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

Binding Sites↗
PubMed1989

Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme.

A shortened form of the intervening sequence of the self-splicing RNA from Tetrahymena thermophila catalyzes sequence-specific cleavage of RNA. Cleavage site selection involves a base-pairing interaction between the substrate RNA and a binding site within the intervening sequence. Single-base changes in this binding site were previously shown to alter substrate specificity in a predictable manner. To examine the generality with which substrate specificity can be altered, six variant catalytic RNAs (ribozymes) have been produced with two- or three-base changes in the active site. Each ribozyme cleaves its predicted substrate. The conditions required for good reactivity and for discrimination against cleavage at mismatched sites vary and were independently determined for each ribozyme.

Animals↗
PubMed1982

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Substrate specificity of the alleloenzymes AdhS and AdhUF.

The substrate specificity of the two alleloenzymes AdhS an AdhUF from Drosophila melanogaster has been studied and found to be similar. With most of the secondary alcohols, the Vm value is essentially the same, and indicative of a Theorell-Chance mechanism with rate-limiting enzyme-coenzyme dissociation. The experiments indicate that the enzyme-coenzyme complex formed with AdhUF dissociates at a faster rate than the corresponding complex with AdhS. For primary alcohols the Vm value is much lower than for secondary alcohols, varies with the type of alcohol and the dissociation of the enzyme-coenzyme complex is not rate limiting. For these alcohols a primary isotope effect with deuteroethanol indicates that it is the interconversion of the ternary complexes that is rate determining. Studies with the enantiomers of butan-2-ol and octan-2-ol show that both alkyl groups in the secondary alcohols interact hydrophobically with the alcohol-binding region of the active site. However, the two parts of the alcohol-binding region that interact with the two alkyl groups are of different size. The high activity observed with secondary alcohols and especially with (R)-(+)-cis-verbenol, indicates that these flies can metabolize terpenes. Such compounds may be part of the pheromone system in the flies with D. melanogaster alcohol dehydrogenase playing a role in pheromone metabolism.

Alcohol Oxidoreductases↗
PubMed1997

A reevaluation of substrate specificity of the rat cation transporter rOCT1.

The substrate specificity of the previously cloned rat cation transporter rOCT1, which is expressed in kidney, liver, and small intestine, was reevaluated. rOCT1 is the first member of a new protein family comprising electrogenic and polyspecific cation transporters that transport hydrophilic cations like tetraethylammonium, choline, and monoamine neurotransmitters. Previous electrical measurements suggested that cations like quinine, quinidine, and cyanine 863, which have been classified as type 2 cations in the liver, are also transported by rOCT1, since they may induce inward currents in rOCT1 expressing Xenopus oocytes (Busch, A. E., Quester, S., Ulzheimer, J. C., Waldegger, S., Gorboulev, V., Arndt, P., Lang, F., and Koepsell, H. (1996) J. Biol. Chem. 271, 32599-32604). Tracer flux measurements with oocytes and with stably transfected human embryonic kidney cells showed that [3H]quinine and [3H]quinidine are not transported by rOCT1. The voltage dependence observed for the quinine- or quinidine-induced inward currents in rOCT1-expressing oocytes, and tracer efflux measurements indicate that the inward currents by type 2 cations are generated by the inhibition of electrogenic efflux of transported type 1 cations. Therefore, rOCT1 cannot contribute to transport of type 2 cations in the liver and the hepatic transporter for type 2 cations remains to be identified.

Animals↗
PubMed1997

Amino acid substitutions that convert the protein substrate specificity of farnesyltransferase to that of geranylgeranyltransferase type I.

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta. Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

Alkyl and Aryl Transferases↗