Recombinant 1-deoxy-D-xylulose-5-phosphate synthase 2 (dxs2), partial

What is 1-deoxy-D-xylulose-5-phosphate synthase 2 (DXS2) and how does it function in the MEP pathway?

1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first enzyme in the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis, which is essential in many bacteria, green algae, and plant chloroplasts. The enzyme catalyzes the decarboxylation of pyruvate and the subsequent condensation with D-glyceraldehyde 3-phosphate (GAP) to form 1-deoxy-D-xylulose 5-phosphate (DXP) . DXS2 represents a second homolog of this enzyme present in some organisms such as Rhodobacter capsulatus and Mycobacterium tuberculosis .

Methodologically, DXS functions as a thiamin diphosphate (ThDP)-dependent enzyme that exhibits unique catalytic properties compared to other ThDP-dependent enzymes. Unlike most enzymes in this family, DXS stabilizes the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which then undergoes decarboxylation triggered by GAP binding .

What expression systems are optimal for producing recombinant DXS2 for research purposes?

Several expression systems have been successfully employed for recombinant DXS production, each with distinct advantages:

When expressing recombinant DXS2, researchers should consider codon optimization for the chosen expression system and the addition of purification tags (e.g., His6-tag) at the N- or C-terminus to facilitate downstream purification . For structural studies, engineered constructs with specific truncations may improve crystallization properties while maintaining enzymatic activity .

How do the kinetic parameters of recombinant DXS enzymes compare between different organisms?

Kinetic analyses of recombinant DXS enzymes from different organisms reveal both similarities and differences in substrate affinity and catalytic efficiency:

These data demonstrate that while DXS enzymes catalyze the same reaction across species, their kinetic properties can vary significantly, which may reflect adaptations to different metabolic environments. For accurate determination of kinetic parameters, researchers should employ standardized assay conditions and consider the influence of buffer components, especially since DXS activity is often measured in sodium citrate buffer (pH 7.4) .

What challenges exist in crystallizing DXS for structural studies, and how can protein engineering address these issues?

Crystallization of DXS has proven particularly challenging, with only a limited number of X-ray structures available despite its significance as a drug target. Key challenges include:

• Flexible regions that hinder crystal formation

• Proteolytic sensitivity during crystallization processes

• Large size (~68-70 kDa per monomer) and complex dimeric structure

• Missing segments in existing crystal structures (residues 183-238 and 292-317 in E. coli DXS)

Recent structural biology approaches have addressed these challenges through protein engineering:

• Truncation strategy: A truncated Deinococcus radiodurans DXS was developed that retains enzymatic activity while improving crystallization properties . This approach targets variable regions without compromising catalytic function.

• In situ proteolysis: Controlled proteolysis during crystallization has facilitated structure determination, though this has resulted in structures with missing segments .

• Anoxic conditions: Recent structures were obtained under anoxic conditions, suggesting oxygen sensitivity may affect crystallization .

Methodologically, researchers should consider employing complementary techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) alongside crystallography to gain insights into regions not resolved in crystal structures, as demonstrated with E. coli DXS where HDX-MS revealed conformational dynamics in segments absent from crystal structures .

How does conformational dynamics influence the catalytic mechanism of DXS?

HDX-MS studies have revealed significant conformational dynamics in DXS that play a crucial role in its unique catalytic mechanism:

• Three regions of E. coli DXS (residues 42-58, 183-199, and 278-298) near the active center display both EX1 (monomolecular) and EX2 (bimolecular) H/D exchange kinetic behavior, indicating significant conformational flexibility .

• These regions undergo conformational changes in response to ligand binding:

  • With substrate analog (methyl acetylphosphate) bound: closed conformation favored

  • With GAP or DXP: open conformation induced

• The conformational changes correlate with key mechanistic steps:

  • Closed conformation: critical for stabilization of the LThDP intermediate

  • GAP-induced opening: coincides with decarboxylation of LThDP and product release

This conformational flexibility distinguishes DXS from other ThDP-dependent enzymes and explains its unique ability to stabilize the predecarboxylation intermediate until GAP binding . For researchers investigating DXS catalysis, techniques that can monitor these conformational changes (such as HDX-MS, FRET, or EPR spectroscopy) should be considered essential complementary approaches to static structural studies.

What are the functional differences between DXS1 and DXS2 in organisms that possess both enzymes?

In organisms with both DXS1 and DXS2, these paralogs exhibit distinct functional characteristics:

• Essentiality: In M. tuberculosis, DXS1 (encoded by dxs1) is essential for growth in vitro, while the second homolog (DXS2, encoded by dxs2) cannot compensate for the loss of DXS1 .

• Expression regulation: DXS1 in M. tuberculosis appears to regulate the expression of the dxr operon (containing dxr and gcpE), indicating a regulatory role beyond its catalytic function .

• Metabolic impact: Overexpression of either DXS1 or DXS2 in M. tuberculosis inhibits growth, likely due to pyruvate depletion, while overexpression of downstream enzymes (Dxr or GcpE) does not show this effect .

• Pathway flux: Overexpression of dxs1 or gcpE in M. tuberculosis increases flux through the MEP pathway, as measured by accumulation of 4-hydroxy-3-methyl-but-2-enyl pyrophosphate .

In R. capsulatus, one DXS gene (dxsA) is located in the photosynthesis gene cluster, while the other (dxsB) is located elsewhere in the chromosome, suggesting specialized roles related to photosynthetic metabolism . Complementation studies have shown that R. capsulatus dxsB can functionally replace the essential E. coli dxs gene in a mutant strain .

These differences suggest that while both enzymes catalyze the same reaction, they likely serve distinct metabolic roles, potentially related to different metabolic demands or environmental conditions.

What methods can be employed to assess the activity of recombinant DXS2?

Several methodological approaches can be utilized to assess DXS activity:

• Coupled enzymatic assays: Monitor the formation of DXP by coupling with subsequent enzymatic reactions that produce detectable products (e.g., colorimetric or fluorescent).

• Direct product detection: Measure DXP formation directly using HPLC or LC-MS/MS methods, which offer high specificity but require specialized equipment.

• Radioactive assays: Use 14C-labeled pyruvate to measure the incorporation of radioactivity into DXP, offering high sensitivity.

• Alternative substrate utilization: DXS can use D-glyceraldehyde instead of GAP as a substrate, though with lower efficiency (Km value of 35 mM for D-glyceraldehyde compared to μM range for GAP) , providing an alternative assay approach.

• Complementation studies: Functionality can be assessed through the ability of the recombinant enzyme to complement an E. coli strain with a disrupted chromosomal dxs gene (e.g., strain FH11) .

When assessing DXS2 activity, researchers should consider that optimal conditions may include pH ~9.0 (as determined for Streptomyces DXS) and that the enzyme likely functions as a homodimer (~130-140 kDa for the assembled complex) .

How can site-directed mutagenesis be used to investigate the catalytic mechanism of DXS2?

Site-directed mutagenesis represents a powerful approach for elucidating the catalytic mechanism of DXS2:

• ThDP binding site: Mutations in residues coordinating the ThDP cofactor can reveal their roles in cofactor binding and positioning for catalysis.

• Substrate binding residues: Altering residues involved in pyruvate or GAP binding can help identify their contributions to substrate specificity and binding affinity.

• Catalytic residues: Mutations of putative catalytic residues can provide insights into their roles in acid-base chemistry during catalysis.

• Conformationally dynamic regions: Targeting the three regions displaying EX1 kinetics (residues 42-58, 183-199, and 278-298 in E. coli DXS) can help assess their functional importance in catalysis.

• Interface residues: As DXS functions as a dimer, mutations at the dimer interface can reveal the importance of dimerization for catalysis.

Methodologically, researchers should employ a combination of structural information, sequence conservation analysis, and computational predictions to guide mutation selection. Mutagenesis can be performed using standard techniques such as Kunkel mutagenesis, which has been successfully applied to introduce modifications in DXS constructs . The impact of mutations should be assessed through multiple parameters including enzyme kinetics, protein stability, oligomeric state, and substrate binding.

What purification strategies are most effective for obtaining high-quality recombinant DXS2?

Efficient purification of recombinant DXS2 typically involves a multi-step approach:

For R. capsulatus DXS proteins containing a C-terminal His6-tag, a two-step purification process achieving >95% homogeneity has been reported . The addition of glycerol (10-20%) to buffers can enhance protein stability, and the inclusion of reducing agents such as DTT or TCEP may be necessary to prevent oxidation of cysteine residues .

Researchers should verify the quality of the purified protein through multiple analytical methods including SDS-PAGE, western blotting, dynamic light scattering (to assess aggregation), and activity assays. For structural studies, additional quality control steps such as thermal shift assays or limited proteolysis may be beneficial to assess conformational homogeneity.

How does the MEP pathway containing DXS2 represent a potential antimicrobial target?

The MEP pathway represents an attractive antimicrobial target due to several key characteristics:

• Pathway essentiality: DXS catalyzes the first and rate-limiting step of the MEP pathway, which is essential in many bacteria including pathogens such as M. tuberculosis .

• Absence in mammals: Mammals utilize the distinct mevalonate pathway for isoprenoid biosynthesis, providing an opportunity for selective targeting without affecting host metabolism .

• Structural uniqueness: DXS possesses unique structural features compared to other ThDP-dependent enzymes, allowing for the potential development of highly specific inhibitors .

• Validation through genetic studies: Genetic disruption studies have demonstrated that the MEP pathway enzymes, including DXS, are essential for bacterial growth and cannot be complemented by alternative pathways .

Research approaches to exploit this target include:

• Structure-based drug design targeting DXS active sites or unique conformational states

• Fragment-based screening to identify novel scaffold inhibitors

• High-throughput screening of compound libraries against recombinant DXS

• Rational design of substrate analogs as competitive inhibitors

Recent developments have included the engineering of a truncated D. radiodurans DXS that retains enzymatic activity while showing improved crystallization properties, facilitating structure-based drug design efforts . This approach could potentially be transferred to DXS homologs from pathogenic species, expanding opportunities for antimicrobial development.

What role do the regions displaying EX1 kinetics play in the function of DXS?

Three regions of E. coli DXS (spanning residues 42-58, 183-199, and 278-298) near the active center display EX1 (monomolecular) H/D exchange kinetic behavior, indicating significant conformational dynamics . These regions play critical functional roles:

• Substrate-induced conformational changes: These regions undergo conformational transitions between open and closed states in response to different ligands:

  • In the absence of ligands: Both conformations exist in equilibrium

  • With pyruvate analog (methyl acetylphosphate): Closed conformation favored

  • With GAP or DXP: Open conformation induced

• Catalytic significance: These conformational changes are essential for the unique catalytic mechanism of DXS:

  • Closed conformation stabilizes the LThDP intermediate

  • GAP binding induces opening, facilitating decarboxylation and product formation

• Structural context: Two of these regions (183-199 and part of 278-298) are missing from crystal structures, suggesting their high flexibility is important for function but challenging for structural studies .

The close spatial proximity of these regions to each other and to the active site suggests their conformational changes occur simultaneously and are coordinated during catalysis . These findings highlight the importance of protein dynamics in DXS function and suggest that targeting these conformationally dynamic regions may present a novel approach for inhibitor design.

How do truncated forms of DXS compare functionally to full-length enzymes?

Truncated forms of DXS have been developed primarily to improve crystallization properties while maintaining enzymatic function. The functional comparisons reveal:

• Catalytic activity: A truncated D. radiodurans DXS retains enzymatic activity despite removal of certain regions, demonstrating that not all segments are essential for basic catalytic function .

• Stability improvements: Truncated forms often show reduced proteolytic degradation, improving protein stability during purification and crystallization processes .

• Structural insights: Truncated variants have facilitated the determination of crystal structures, providing valuable structural information that was previously unobtainable with full-length proteins .

• Target regions: The truncation sites typically target variable regions rather than highly conserved domains, preserving the core catalytic machinery .

The development of functional truncated forms has significant implications for structural biology and drug discovery efforts targeting DXS. The identification of regions that can be removed without compromising function suggests these segments may play roles in regulation, protein-protein interactions, or adaptation to specific cellular environments rather than being directly involved in catalysis.

Methodologically, when developing truncated forms of DXS2, researchers should consider evolutionary conservation analysis, secondary structure predictions, and available structural information to guide truncation site selection. Activity assays and stability studies should be conducted to confirm the functionality of the engineered constructs.

What complementation studies have been conducted to evaluate the functional interchangeability of DXS1 and DXS2?

Several complementation studies have provided insights into the functional relationships between DXS homologs:

• R. capsulatus DXS complementation: The dxsB gene from R. capsulatus has been shown to complement E. coli strain FH11, in which the chromosomal copy of dxs is disrupted . This demonstrates that R. capsulatus DXS-B is functionally capable of replacing E. coli DXS.

• M. tuberculosis DXS homologs: Studies in M. tuberculosis have demonstrated that DXS1 is essential and that DXS2 cannot compensate for its loss, indicating functional differences between these paralogs .

• Cross-species complementation: E. coli FH11 (with disrupted chromosomal dxs) can also be complemented by growth on 2-methylerythritol or mevalonate when transformed with a plasmid containing yeast genes required for IPP biosynthesis from mevalonate . This demonstrates the potential for alternative pathway complementation.

These studies reveal the complex nature of functional conservation and divergence among DXS homologs. While some homologs retain sufficient functional conservation to complement across species, paralogs within the same organism may have diverged to serve specialized metabolic roles that prevent functional interchangeability.

Methodologically, complementation studies typically involve generating conditional or deletion mutants and introducing plasmid-expressed homologs to assess growth rescue. Such approaches provide valuable insights into functional relationships that may not be apparent from sequence analysis or in vitro studies alone.

How can recombinant DXS2 be optimized for expression yield and solubility?

Optimization of recombinant DXS2 expression requires addressing several key factors:

For DXS specifically, the addition of a glycine and six histidine residues at the C-terminus has been successfully employed for R. capsulatus DXS expression . Additionally, the use of protease-deficient host strains may be beneficial given the potential sensitivity of DXS to proteolytic degradation .

When engineering constructs, researchers should consider that DXS functions as a dimer, so modifications should not disrupt the dimerization interface. Thermal shift assays can be valuable for identifying buffer conditions that maximize protein stability, potentially improving both yield and downstream applications.

What analytical techniques are most effective for characterizing the structural properties of recombinant DXS2?

A comprehensive structural characterization of recombinant DXS2 requires a multi-technique approach:

For DXS specifically, combining techniques that capture both static structure (crystallography) and dynamic behavior (HDX-MS) has proven particularly informative for understanding the enzyme's unique catalytic mechanism and conformational changes . Researchers should consider that DXS undergoes significant conformational changes during catalysis, so characterization in various ligand-bound states is essential for a complete structural understanding.

Frequently Asked Questions for Researchers: Recombinant 1-deoxy-D-xylulose-5-phosphate synthase 2 (dxs2), partial

What is 1-deoxy-D-xylulose-5-phosphate synthase 2 (DXS2) and how does it function in the MEP pathway?

1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first enzyme in the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis, which is essential in many bacteria, green algae, and plant chloroplasts. The enzyme catalyzes the decarboxylation of pyruvate and the subsequent condensation with D-glyceraldehyde 3-phosphate (GAP) to form 1-deoxy-D-xylulose 5-phosphate (DXP) . DXS2 represents a second homolog of this enzyme present in some organisms such as Rhodobacter capsulatus and Mycobacterium tuberculosis .

Methodologically, DXS functions as a thiamin diphosphate (ThDP)-dependent enzyme that exhibits unique catalytic properties compared to other ThDP-dependent enzymes. Unlike most enzymes in this family, DXS stabilizes the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which then undergoes decarboxylation triggered by GAP binding .

What expression systems are optimal for producing recombinant DXS2 for research purposes?

Several expression systems have been successfully employed for recombinant DXS production, each with distinct advantages:

When expressing recombinant DXS2, researchers should consider codon optimization for the chosen expression system and the addition of purification tags (e.g., His6-tag) at the N- or C-terminus to facilitate downstream purification . For structural studies, engineered constructs with specific truncations may improve crystallization properties while maintaining enzymatic activity .

How do the kinetic parameters of recombinant DXS enzymes compare between different organisms?

Kinetic analyses of recombinant DXS enzymes from different organisms reveal both similarities and differences in substrate affinity and catalytic efficiency:

These data demonstrate that while DXS enzymes catalyze the same reaction across species, their kinetic properties can vary significantly, which may reflect adaptations to different metabolic environments. For accurate determination of kinetic parameters, researchers should employ standardized assay conditions and consider the influence of buffer components, especially since DXS activity is often measured in sodium citrate buffer (pH 7.4) .

What challenges exist in crystallizing DXS for structural studies, and how can protein engineering address these issues?

Crystallization of DXS has proven particularly challenging, with only a limited number of X-ray structures available despite its significance as a drug target. Key challenges include:

• Flexible regions that hinder crystal formation

• Proteolytic sensitivity during crystallization processes

• Large size (~68-70 kDa per monomer) and complex dimeric structure

• Missing segments in existing crystal structures (residues 183-238 and 292-317 in E. coli DXS)

Recent structural biology approaches have addressed these challenges through protein engineering:

• Truncation strategy: A truncated Deinococcus radiodurans DXS was developed that retains enzymatic activity while improving crystallization properties . This approach targets variable regions without compromising catalytic function.

• In situ proteolysis: Controlled proteolysis during crystallization has facilitated structure determination, though this has resulted in structures with missing segments .

• Anoxic conditions: Recent structures were obtained under anoxic conditions, suggesting oxygen sensitivity may affect crystallization .

Methodologically, researchers should consider employing complementary techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) alongside crystallography to gain insights into regions not resolved in crystal structures, as demonstrated with E. coli DXS where HDX-MS revealed conformational dynamics in segments absent from crystal structures .

How does conformational dynamics influence the catalytic mechanism of DXS?

HDX-MS studies have revealed significant conformational dynamics in DXS that play a crucial role in its unique catalytic mechanism:

• Three regions of E. coli DXS (residues 42-58, 183-199, and 278-298) near the active center display both EX1 (monomolecular) and EX2 (bimolecular) H/D exchange kinetic behavior, indicating significant conformational flexibility .

• These regions undergo conformational changes in response to ligand binding:

  • With substrate analog (methyl acetylphosphate) bound: closed conformation favored

  • With GAP or DXP: open conformation induced

• The conformational changes correlate with key mechanistic steps:

  • Closed conformation: critical for stabilization of the LThDP intermediate

  • GAP-induced opening: coincides with decarboxylation of LThDP and product release

This conformational flexibility distinguishes DXS from other ThDP-dependent enzymes and explains its unique ability to stabilize the predecarboxylation intermediate until GAP binding . For researchers investigating DXS catalysis, techniques that can monitor these conformational changes (such as HDX-MS, FRET, or EPR spectroscopy) should be considered essential complementary approaches to static structural studies.

What are the functional differences between DXS1 and DXS2 in organisms that possess both enzymes?

In organisms with both DXS1 and DXS2, these paralogs exhibit distinct functional characteristics:

• Essentiality: In M. tuberculosis, DXS1 (encoded by dxs1) is essential for growth in vitro, while the second homolog (DXS2, encoded by dxs2) cannot compensate for the loss of DXS1 .

• Expression regulation: DXS1 in M. tuberculosis appears to regulate the expression of the dxr operon (containing dxr and gcpE), indicating a regulatory role beyond its catalytic function .

• Metabolic impact: Overexpression of either DXS1 or DXS2 in M. tuberculosis inhibits growth, likely due to pyruvate depletion, while overexpression of downstream enzymes (Dxr or GcpE) does not show this effect .

• Pathway flux: Overexpression of dxs1 or gcpE in M. tuberculosis increases flux through the MEP pathway, as measured by accumulation of 4-hydroxy-3-methyl-but-2-enyl pyrophosphate .

In R. capsulatus, one DXS gene (dxsA) is located in the photosynthesis gene cluster, while the other (dxsB) is located elsewhere in the chromosome, suggesting specialized roles related to photosynthetic metabolism . Complementation studies have shown that R. capsulatus dxsB can functionally replace the essential E. coli dxs gene in a mutant strain .

These differences suggest that while both enzymes catalyze the same reaction, they likely serve distinct metabolic roles, potentially related to different metabolic demands or environmental conditions.

What methods can be employed to assess the activity of recombinant DXS2?

Several methodological approaches can be utilized to assess DXS activity:

• Coupled enzymatic assays: Monitor the formation of DXP by coupling with subsequent enzymatic reactions that produce detectable products (e.g., colorimetric or fluorescent).

• Direct product detection: Measure DXP formation directly using HPLC or LC-MS/MS methods, which offer high specificity but require specialized equipment.

• Radioactive assays: Use 14C-labeled pyruvate to measure the incorporation of radioactivity into DXP, offering high sensitivity.

• Alternative substrate utilization: DXS can use D-glyceraldehyde instead of GAP as a substrate, though with lower efficiency (Km value of 35 mM for D-glyceraldehyde compared to μM range for GAP) , providing an alternative assay approach.

• Complementation studies: Functionality can be assessed through the ability of the recombinant enzyme to complement an E. coli strain with a disrupted chromosomal dxs gene (e.g., strain FH11) .

When assessing DXS2 activity, researchers should consider that optimal conditions may include pH ~9.0 (as determined for Streptomyces DXS) and that the enzyme likely functions as a homodimer (~130-140 kDa for the assembled complex) .

How can site-directed mutagenesis be used to investigate the catalytic mechanism of DXS2?

Site-directed mutagenesis represents a powerful approach for elucidating the catalytic mechanism of DXS2:

• ThDP binding site: Mutations in residues coordinating the ThDP cofactor can reveal their roles in cofactor binding and positioning for catalysis.

• Substrate binding residues: Altering residues involved in pyruvate or GAP binding can help identify their contributions to substrate specificity and binding affinity.

• Catalytic residues: Mutations of putative catalytic residues can provide insights into their roles in acid-base chemistry during catalysis.

• Conformationally dynamic regions: Targeting the three regions displaying EX1 kinetics (residues 42-58, 183-199, and 278-298 in E. coli DXS) can help assess their functional importance in catalysis.

• Interface residues: As DXS functions as a dimer, mutations at the dimer interface can reveal the importance of dimerization for catalysis.

Methodologically, researchers should employ a combination of structural information, sequence conservation analysis, and computational predictions to guide mutation selection. Mutagenesis can be performed using standard techniques such as Kunkel mutagenesis, which has been successfully applied to introduce modifications in DXS constructs . The impact of mutations should be assessed through multiple parameters including enzyme kinetics, protein stability, oligomeric state, and substrate binding.

What purification strategies are most effective for obtaining high-quality recombinant DXS2?

Efficient purification of recombinant DXS2 typically involves a multi-step approach:

For R. capsulatus DXS proteins containing a C-terminal His6-tag, a two-step purification process achieving >95% homogeneity has been reported . The addition of glycerol (10-20%) to buffers can enhance protein stability, and the inclusion of reducing agents such as DTT or TCEP may be necessary to prevent oxidation of cysteine residues .

Researchers should verify the quality of the purified protein through multiple analytical methods including SDS-PAGE, western blotting, dynamic light scattering (to assess aggregation), and activity assays. For structural studies, additional quality control steps such as thermal shift assays or limited proteolysis may be beneficial to assess conformational homogeneity.

How does the MEP pathway containing DXS2 represent a potential antimicrobial target?

The MEP pathway represents an attractive antimicrobial target due to several key characteristics:

• Pathway essentiality: DXS catalyzes the first and rate-limiting step of the MEP pathway, which is essential in many bacteria including pathogens such as M. tuberculosis .

• Absence in mammals: Mammals utilize the distinct mevalonate pathway for isoprenoid biosynthesis, providing an opportunity for selective targeting without affecting host metabolism .

• Structural uniqueness: DXS possesses unique structural features compared to other ThDP-dependent enzymes, allowing for the potential development of highly specific inhibitors .

• Validation through genetic studies: Genetic disruption studies have demonstrated that the MEP pathway enzymes, including DXS, are essential for bacterial growth and cannot be complemented by alternative pathways .

Research approaches to exploit this target include:

• Structure-based drug design targeting DXS active sites or unique conformational states

• Fragment-based screening to identify novel scaffold inhibitors

• High-throughput screening of compound libraries against recombinant DXS

• Rational design of substrate analogs as competitive inhibitors