Kinetic Study of the Effect of Histidines 240 and 164 on TEM-149 Enzyme Probed by β-Lactam Inhibitors

Abstract

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149^H240, and TEM-149^H164-H240 versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149^H240, and TEM-149^H164-H240 β-lactamases, and the K_i (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k₊₂ and k₊₃ (first-order acylation and deacylation constants, respectively), and k₊₂/K values were calculated.

TEXT

TEM-1 is the prototype of the TEM family of enzymes, which can hydrolyze penicillins and cephalosporins. During the last 20 years, TEM-1 underwent a natural evolution because of the selective pressure of β-lactams (1). Several TEM variants, including extended-spectrum β-lactamases (ESBLs) and inhibitor-resistant TEMs (IRTs), have been generated (217 TEM enzymes by K. Bush and G. A. Jacoby [http://www.lahey.org/Studies/]). Such high numbers of variants are the consequence of the ability of the TEM β-lactamases to tolerate amino acid substitutions. The plasticity of these enzymes is increased by their ability to acquire compensatory mutations. Nevertheless, only a few mutations are directly involved in enzymatic catalysis. Some of these mutations expand the ESBL phenotype, while others are involved in enzyme stability as M182T and L201P substitutions (2).

TEM-149, an extended-spectrum β-lactamase isolated in Italy in 2003 from Enterobacter aerogenes and Serratia marcescens, shows an array of amino acid changes, including E104K, R164S, M182T, and E240V (3). The novelty of this enzyme is the unexpected presence of valine at position 240 in place of the common glutamic acid or lysine. As previously described, the combination of mutations found in TEM-149 enhances catalytic efficiency versus that from ceftazidime and aztreonam. This behavior is most probably due to a better accommodation of these substrates in the catalytic site of the enzyme (3).

The production and purification of the TEM-149, TEM-149^H240, and TEM-149^H164-H240 enzymes were performed as described elsewhere (3, 4).

Nitrocefin was kindly provided by Shariar Mobashery (Notre Dame University, South Bend, IN, USA). Meropenem was from AstraZeneca (Milan, Italy). Imipenem, ertapenem, and cefoxitin were from Merck Sharp & Dohme (Rome, Italy). Temocillin, ticarcillin, and BRL42715b were from SmithKline Beecham (Brentford, United Kingdom). Tigemonam was from Bristol-Myers Squibb (New York, NY, USA). Carumonam was from Roche (Milan, Italy). The penem CP65207 was from Pfizer (New York, NY, USA). The penem HR664 was from Hoechst Italia (Milan, Italy). Biapenem was from Cyanamid (Catania, Italy). Loracarbef (carbacephem) was from Eli Lilly and Co. (Indianapolis, IN, USA). Most of these compounds, such as BRL42715b, CP65207, and HR664, were conserved in our laboratory for long periods of time under appropriate conditions, and their stabilities and concentrations were verified by spectrophotometric analysis.

Steady-state kinetic experiments were carried out at 25°C in 50 mM sodium phosphate buffer (pH 7.0) containing 0.2 M KCl to preserve enzyme stability. Inhibition assays were performed using nitrocefin as a substrate reporter at concentrations that ranged from 50 to 100 μM.

Competitive inhibition assays were performed under the following conditions:

$$v_0/v_i=\text{1}+(K_m\times I)/\left(K_m+S\right)\times K_i$$ (1)

where v_i and v₀ represent the initial rates of hydrolysis of nitrocefin with or without inhibitor, respectively, I is the concentration of the inhibitor or poor substrate, K_i is the inhibition constant, K_m is the Henri-Michaelis constant, and S is the concentration of the reporter substrate. The plot of v₀/v_i versus [I] yielded a straight line of slope K_m/(K_m + S) × K_i (5).

In compounds behaving as transient inhibitors, the accumulation and slow hydrolysis of E·C* were studied on the basis of the following model:

$$E+C\hspace{0.17em}\stackrel{K}{\rightleftarrows }\hspace{0.17em}E·C\hspace{0.17em}\stackrel{k_{+2}}{\to }\hspace{0.17em}EC*\hspace{0.17em}\stackrel{k_{+3}}{\to }\hspace{0.17em}E+P$$ (2)

where E is the enzyme, C is the substrate, E·C is the Henri-Michaelis complex, EC* is the acyl-enzyme complex, and P is the hydrolysis product. k₊₂ and k₊₃ are the first-order acylation and deacylation constants, respectively. K is the dissociation constant of the Henri-Michaelis complex.

The values of k_i (first-order rate constant which characterized the EC* accumulation) were obtained by time course hydrolysis of nitrocefin following equations 2 and 3, as previously determined (5, 6):

$$\left(v_t–v_{ss}\right)/\left(v_0–v_{ss}\right)=e^{–kit}$$ (3)

where v₀, v_t, and v_ss are the rate transformations of the substrate at time zero, at time t, and at steady state:

$$k_i=k_{+3}+\frac{k_{+2}\cdot [C]}{[C]+K\cdot \left(1+[S]/K_{m}S\right)}$$ (4)

where [S] is the concentration of the reporter substrate, and K_mS is the K_m of the reporter substrate. The condition [S] ≈ K_m, where [S] is the concentration of the substrate reporter, was respected.

When k_i varied linearly with [C] (indicating that the range of [C] was ≪K), then the k₊₂/K value was calculated from the slope of the line, and k₊₃ was obtained from the extrapolation, with [C] equal to 0.

In the case of inactivator BRL42715b, the plot of k_i versus [C] was not linear (indicating that [C] was ≥K), and k₊₂ and K were calculated by plotting [C]/k_i versus [C] (5, 6).

Thirteen compounds, including penicillin, penems, carbapenems, monobactams, carbacephem, and cephamycin, were tested as molecules able to inhibit or inactivate the enzymes TEM-149, TEM-149^H240 (single mutant), and TEM-149^H164-H240 (double mutant). Tables 1 and 2 summarize the values of K_i, K, k₊₂, k₊₃, and k₊₂/K calculated for the three enzymes. All the tested compounds behaved as poor substrates versus the three enzymes.

TABLE 1.

Compounds that behave as competitive inhibitors

Inhibitor	Ki (μM) for:
	TEM-149	TEM-149H240	TEM-149H164-H240
Temocillin (penicillin)	8.30	13.5	260.00
Ticarcillin (carboxypenicillin)	NDa	2.50	ND
HR664 (penem)	14.00	32.00	ND
CP65207 (penem)	ND	11.00	41.00
BRL42715b (penem)	ND	0.36	ND
Carumonam (monobactam)	2.00	7.10	>3,000
Tigemonam (monobactam)	0.0048	0.029	207.00
Loracarbef (carbacephem)	150.00	38.00	440.00

^aND, not determined.

TABLE 2.

Compounds that behave as transient inactivators

Inactivator	TEM-149		TEM-149H240		TEM-149H164-H240		k+2 (s−1)	K (μM)
	k+3 (s−1)	k+2/K (μM−1 s−1)	k+3 (s−1)	k+2/K (μM−1 s−1)	k+3 (s−1)	k+2/K (μM−1 s−1)
Meropenem	2.00 × 10−2	1.10 × 10−3	2.31 × 10−2	0.40 × 10−3	1.98 × 10−2	0.40 × 10−3	NDa	ND
Imipenem	2.66 × 10−2	0.80 × 10−3	1.65 × 10−2	0.50 × 10−3	2.44 × 10−2	0.40 × 10−3	ND	ND
Ertapenem	1.67 × 10−2	12.00 × 10−3	1.60 × 10−2	5.20 × 10−3	1.80 × 10−3	1.40 × 10−3	ND	ND
Biapenem	0.80 × 10−2	2.50 × 10−3	0.38 × 10−2	2.30 × 10−3	1.90 × 10−2	1.20 × 10−3	ND	ND
HR664	ND	ND	ND	ND	1.50 × 10−2	0.30 × 10−3	ND	ND
CP65207	1.75 × 10−2	0.80 × 10−3	ND	ND	ND	ND	ND	ND
BRL42715b	1.55 × 10−2	0.35 × 10−3	ND	ND	0	3.30	6.77	59
Cefoxitin	1.74 × 10−2	2.00 × 10−5	0.69 × 10−2	2.00 × 10−5	ND	ND	ND	ND
Ticarcillin	0.92 × 10−2	9.00 × 10−4	ND	ND	2.31 × 10−2	0.20 × 10−3	ND	ND

^aND, not determined.

Ticarcillin and temocillin.

Ticarcillin behaved as a competitive inhibitor for TEM-149^H240, with a K_i value of 2.5 μM (Table 1), whereas it can be considered a transient inactivator, with k₊₃ not equal to 0 for TEM-149 and TEM-149^H164-H240. The k₊₃ value calculated for the double mutant was about 2.5-fold higher than the k₊₃ of TEM-149. The k₊₂/K for the double mutant was 4-fold lower than that calculated for TEM-149. Temocillin, a 6α-methoxy β-lactam derivative of ticarcillin, seemed to behave similarly for the three enzymes. For instance, it can be considered a competitive inhibitor, with K_i values of 8.3, 13.5, and 260 μM for TEM-149, TEM-149^H240, and TEM-149^H164-H240, respectively (Table 1). The K_i values calculated for TEM-149 and the single mutant were similar, while the double mutant showed a reduced affinity.

Penems.

In the present study, the penems used for kinetic and physical studies of β-lactamase inhibition (7–9) also were tested in order to highlight minimal differences between TEM-149 and TEM-149^H240/TEM-149^H164-H240.

The HR664 compound acted as a reversible competitive inhibitor for TEM-149 and TEM-149^H240 but as a transient inactivator with a measurable turnover (k₊₃ ≠ 0) for TEM-149^H164-H240.

The penem CP65207 behaved as a transient inactivator toward the TEM-149 enzyme but was a competitive inhibitor of TEM-149^H240 and TEM-149^H164-H240. BRL42715b showed different behaviors when tested against the three enzymes. It behaved as a reversible competitive inhibitor versus TEM-149^H240, with a K_i of 0.36 μM (Table 1); it was a transient inactivator for TEM-149, with k₊₃ not equal to 0 (Table 2), and an inactivator for TEM-149^H164-H240, with k₊₃ equal to 0 (Table 2).

Carbapenems.

The k₊₃ and k₊₂/K values calculated for all three enzymes versus carbapenems were similar. Ertapenem showed k₊₂/K values for single and double mutants of about 9-fold and 2.5-fold lower than the same value calculated for the TEM-149 enzyme. In contrast, the k₊₃ values were comparable for all three enzymes (Table 2).

Monobactams.

As previously reported by Perilli et al. (4), aztreonam was a good substrate for TEM-149, TEM-149^H240, and TEM-149^H164-H240. On the contrary, carumonam and tigemonam were very poor substrates and can be considered competitive inhibitors versus the three enzymes. The double mutant TEM-149^H164-H240 showed a considerable reduction of the affinity (K_i ≫ 3 mM). Furthermore, tigemonam was a good competitive inhibitor (Table 2).

Cefoxitin and loracarbef.

Cefoxitin acted as a transient inactivator, with k₊₃ not equal to 0 for TEM-149 and TEM-149^H240. For TEM-149^H164-H240, a cefoxitin concentration of up to 3 mM did not inhibit/inactivate the enzyme. In this case, the simultaneous presence of H164 and H240 decreased the affinity of the enzyme for cefoxitin. Carbacephem (Loracarbef) was a competitive inhibitor toward all three enzymes. The single mutant showed a K_i value 14-fold lower than that of the double mutant.

TEM-149 is an ESBL with an unusual valine residue at position 240. In TEM variants, position 240 is generally occupied by a glutamic acid residue and, in many variants (27 clinical isolates and 17 laboratory mutants), by a lysine residue. In our previous study, V240 was replaced by a residue of histidine to generate the single mutant TEM-149^H240, and S164 was replaced by histidine to generate the double mutant TEM-149^H164-H240 (4). The valine residue at position 240 was first found in TEM-149 (3) and in one laboratory mutant (10). Histidine at position 164 has been found in 19 clinical isolates (11) and in 17 laboratory mutants (10). In contrast, the histidine at position 240 was found in only two laboratory mutants generated by antibiotic selective pressure (10). Site-directed mutagenesis on amino acid residues that are generally not found in clinical isolates helps us understand the role of some noncatalytic residues in substrate or inhibitor activity. The presence of a histidine residue at position 240 in the TEM-149^H240 and TEM-149^H164-H240 enzymes decreases their affinities for the tested compounds with respect to TEM-149. As previously indicated, the enzymes TEM-149, TEM-149^H240, and TEM-149^H164-H240 show different behaviors only versus ceftazidime (4). In the present study, using a large panel of molecules with relevant differences near the β-lactam nucleus, we demonstrated that the introduction of one or two histidines in the catalytic pocket of TEM-149 drastically changed the affinities of the mutants. In particular, BRL42715b, HR664, and cefoxitin allowed diversification of single and double mutants from TEM-149. Indeed, each enzyme showed a different kinetic model. Penem BRL42715b can be considered an effective inhibitor of class A, C, and D β-lactamases (6, 7, 12) and a good substrate for metallo-β-lactamases (7). In addition, old penems have been useful in discriminating the activities of the three enzymes. TEM enzymes are good models for these studies because of their plasticity. They provide a good template for designing new drugs and/or inhibitors.