A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori {alpha}1,3/4 fucosyltransferase determines substrate specificity

Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893-21900) demonstrated that exchange of the hypervariable loops, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.     

Characterization of rat and mouse NAD+-dependent 3alpha/17beta/20alpha-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis

In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD⁺ as the preferred coenzyme, and showed low 3α/17β/20α-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD⁺-dependent 3α-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3α-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3α-HSD activity and appearance of 17β- and 20α-HSD activities. AKR1C16 is also 92% identical with rat NAD⁺-dependent 17β-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phe129 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17β- and 20α-hydroxysteroids.     

Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis.

Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.     

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3^– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

C-terminal amino acids of Helicobacter pylori alpha1,3/4 fucosyltransferases determine type I and type II transfer

The alpha1,3/4 fucosyltransferase (FucT) enzyme from Helicobacter pylori catalyzes fucose transfer from donor GDP-beta-l-fucose to the GlcNAc group of two series of acceptor substrates in H. pylori lipopolysaccharide: betaGal1,3betaGlcNAc (Type I) or betaGal1,4betaGlcNAc (Type II). Fucose is added either in alpha1,3 linkage of Type II acceptor to produce Lewis X or in alpha1,4 linkage of Type I acceptor to produce Lewis A, respectively. H. pylori FucTs from different strains have distinct Type I or Type II substrate specificities. FucT in H. pylori strain NCTC11639 has an exclusive alpha1,3 activity because it recognizes only Type II substrates, whereas FucT in H. pylori strain UA948 can utilize both Type II and Type I acceptors; thus it has both alpha1,3 and alpha1,4 activity, respectively. To identify elements conferring substrate specificity, 12 chimeric FucTs were constructed by domain swapping between 11639FucT and UA948FucT and characterized for their ability to transfer fucose to Type I and Type II acceptors. Our results indicate that the C-terminal region of H. pylori FucTs controls Type I and Type II acceptor specificity. In particular, the highly divergent C-terminal portion, seven amino acids DNPFIFC at positions 347-353 in 11639FucT, and the corresponding 10 amino acids CNDAHYSALH at positions 345-354 in UA948FucT, controls the Type I and Type II acceptor recognition. This is the opposite of mammalian FucTs where acceptor preference is determined primarily by the N-terminal residues in the hypervariable stem domain.     

Nucleotide substitution in the amino acid acceptor stem of lysine transfer RNA causes missense suppression

Previous results from this laboratory indicated that, in Escherichia coli K12, a new class of missense suppressors, which read the lysine codons AAA and AAG, may be misacylated lysine transfer RNAs. We therefore isolated and determined the nucleotide sequence of the lysine tRNA from two of the suppressor strains. In each case, we found both wild-type and mutant species of lysine tRNA, a result consistent with evidence that there are two genes for lysine tRNA in the E coli genome. The wild-type sequence was essentially identical to that reported for lysine tRNA from E. coli B. The mutant species isolated from each suppressor strain had a U for C70 nucleotide substitution, demonstrating that the AAG suppressor is a mutant lysine tRNA. The nucleotide substitution in the amino acid acceptor stem is consistent with the in vivo evidence that the suppressor corrects AAA and AAG missense mutations by inserting an amino acid other than lysine during polypeptide synthesis. This report represents the first verification of missense suppression caused by misacylation of a mutant tRNA.     

Structure/function study of Lewis alpha3- and alpha3/4-fucosyltransferases: the alpha1,4 fucosylation requires an aromatic residue in the acceptor-binding domain

All vertebrate alpha3- and alpha3/4-FUTs possess the characteristic acceptor-binding motif VxxHH(W/R)(D/E). FUT6 and FUTb enzymes, harboring R in the acceptor-binding motif, transfer fucose in alpha1,3 linkage, whereas FUT3 and FUT5 enzymes with W at the candidate position can also transfer fucose in alpha1,4 linkage-FUT3 being more efficient than FUT5. To determine the involvement of the W/R residue in acceptor recognition, we produced 34 variants of human FUT3, FUT5, FUT6, and ox FUTb Lewis enzymes. Among the FUT3 variants where W(111) was replaced by the other amino acids, only enzymes with an aromatic residue at the candidate position kept about 50% of alpha1,4 activity and showed no changes in K(m) values for GDP-Fuc donor and H-type 1 acceptor substrates. All other substitutions produced enzymes with less than 20% of the alpha1,4 activity. Thus the ability of alpha3/4-FUTs to recognize type 1 substrates involves the aromatic character of W in the acceptor-binding domain. The alpha1,3 activity of FUT6 and FUTb significantly decreased when their R residue was substituted by basic or charged residues. Moreover, FUT3 and FUT5 variants with W-->R substitution had a better affinity for H-type 2 substrate and higher alpha1,3 activities. Therefore the optimal fucose addition in alpha1,3 linkage requires the R residue in the acceptor-binding motif of Lewis FUTs.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^? null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine

The salvage pathways of nucleotide biosynthesis are more diverse and are less well understood as compared with de novo pathways. Uridine-cytidine kinase (UCK) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. In this study, we have characterized a UCK homologue of Thermus thermophilus HB8 (ttCK) biochemically and structurally. Unlike other UCKs, ttCK had substrate specificity toward only cytidine and showed no inhibition by UTP, suggesting uridine does not bind to ttCK as substrate. Structural analysis revealed that the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK. Replacement of Tyr93 by histidine or glutamine endowed ttCK with phosphorylation activity toward uridine. These results suggested that a single amino acid residue, Tyr93, gives cytidine-limited specificity to ttCK. However, replacement of Tyr93 by Phe or Leu did not change the substrate specificity of ttCK. Therefore, we conclude that a residue at this position is essential for the recognition of uridine by UCK. In addition, thymidine phosphorylase from T. thermophilus HB8 was equally active with thymidine and uridine, which indicates that this protein is the sole enzyme metabolizing uridine in T. Thermophilus HB8. On the basis of these results, we discuss the pyrimidine-salvage pathway in T. thermophilus HB8.     

Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates

Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had approximately 10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.     

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.     

A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.     

Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell-binding domain using site-directed mutagenesis

Previous studies of adhesion mediated by the central cell-binding domain of fibronectin suggest that additional polypeptide information besides the Arg-Gly-Asp sequence is required for full activity. We analyzed this putative second, synergistic region of fibronectin more extensively by deletion analysis and oligonucleotide-based site-directed mutagenesis. Resulting mutated fusion proteins expressed using lambda gt11 were assayed for baby hamster kidney fibroblast cell spreading activity. Deletion mutants truncating from the amino terminus showed a decrease of activity in two apparently discrete steps. Complementary studies using a series of overlapped internal deletions designed to retain the repetitive fibronectin structure also indicated that two distinct peptide regions besides the RGD sequence were necessary for full activity. Removal of the carboxyl-terminal region resulted in the greatest loss of activity (greater than or equal to 20- versus 3-5-fold). Very similar results were obtained with HT-1080 cells dependent on the alpha 5 beta 1 integrin receptor for adhesion to fibronectin. An anti-fibronectin monoclonal antibody that inhibits cell adhesion was found to bind to the carboxyl-terminal functional region, and a point mutation caused specific loss of its epitope. These studies reveal unexpected complexity in the organization of these functional regions, which contrasts with adhesion models based only on simple, short peptide recognition sequences.     

Functional mapping of an entomocidal delta-endotoxin. Single amino acid changes produced by site-directed mutagenesis influence toxicity and specificity of the protein

Mutagenesis has been used to investigate the toxicity and specificity of a larvicidal protein from Bacillus thuringiensis aizawai IC1 that is toxic to both lepidoptera and diptera and differs by only three residues from a monospecific lepidopteran toxin from B. thuringiensis berliner. Site-directed mutagenesis was used to investigate the contribution of these residues to the dual specificity of the aizawai protein. The results suggest that changes in the identity of residues adjacent to Arg544 and Arg567 on the C-terminal side may convert a monospecific toxin into a dual specificity toxin by altering the protease sensitivity of the arginyl peptide bond. A series of deletion mutants was constructed and their protein products analysed for toxicity in vitro and in vivo and for their ability to perturb phospholipid bilayers. The results indicate a different functional role for various protein segments in the toxin's mode of action and suggest that two separate regions close to the C terminus of the active toxin are important in conferring dual specificity on the aizawai IC1 toxin. A model suggesting a basis for the activity of monospecific and dual-specificity B. thuringiensis toxins is presented, which postulates that association of sequences at the C terminus of the active toxin with regions near the N terminus may be responsible for determining toxin specificity.     

A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans.

Summary Gluconobacter oxydans contains pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH). Two isogenic G. oxydans strains, P1 and P2, which differ in their substrate specificity with respect to oxidation of sugars have been analysed. P1 can oxidize only d-glucose, whereas P2 is also capable of the oxidation of the disaccharide maltose. To investigate the nature of this maltose-oxidizing property we cloned the gene encoding GDH from P2. Expression of P2 gdh in P1 enables the latter strain to oxidize maltose, indicating that a mutation in the P2 gdh gene is responsible for the change in substrate specificity. This mutation could be ascribed to a 1 by substitution resulting in the replacement of His 787 by Asn.     

Purification, kinetic characterization, and mapping of the minimal catalytic domain and the key polar groups of Helicobacter pylori alpha-(1,3/1,4)-fucosyltransferases

The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.     

The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem.

Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.