The freedom to try new things and experiments outside the normal scope of the laboratory, together with lively Thursday lunchtime discussions, made for a very good education. I would like to thank all members of the Richards group, past and present, for scientific and other assistance and guidance. Far-flung adventures with members of the Pasadena Mountain Bike Club; The "snow trip" will not be forgotten.
SUMMARY: ~-lactamases are a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolysis of the ~-lactam ring, thereby inactivating the antibiotic. The results of the combinatorial mutagenesis experiments indicate that Lysis is absolutely required for activity at position 73; no mutation at residue 166 can compensate for the loss of the long side chain. The identity of the residue responsible for enhancing the serine at the active site (Ser 70) in RTEM-1 β-lactamase has been disputed for some time.
This indicates that residue 73 does not act as a general base in the acylation step of the reaction. Since the enzyme undergoes conformational changes upon substrate binding, the measured pKa of the free enzyme may not correspond to the pKa of the enzyme-substrate complex.
Chapter 1
Of the three major classes of biological molecules, nucleic acids, fatty acids and proteins, proteins exhibit the widest range of structures and functions (1). Proteins and peptides are polyamides composed of α-amino acids, and most proteins are composed only of a collection of the twenty so-called "natural" amino acids. Amino acids are molecules that contain an amino group, a carboxylic acid group, and another group (R) that determines the identity of the amino acid.
All naturally occurring amino acids are chiral (except glycine, in which R=hydrogen) and of the two possible enantiomers, only the L-enantiomer is found in most peptides and proteins. Most nonpolar amino acids have R groups that are hydrocarbon in nature and may be important in hydrophobic interactions. Many of the polar R groups have ionizable R groups (or side chains), are involved in a variety of interactions, and can function in catalysis (Figure 1).
The nitrogen lone pair is conjugated to the carbonyl double bond as shown in Figure 3, which gives the C-N bond the character of a partial double bond. It is energetically favorable for each of the atoms connected by the C-N bond to lie in the same plane and thus the barrier to rotation is quite high (-80 kJ mol-1) (2).
DACOOH
The substrate in an enzyme-catalyzed reaction binds only to a small area of the enzyme called the active site of the enzyme. Therefore, the entire protein outside the active site can act as a scaffold that holds the active site in the correct geometry for catalysis. Once a substrate binds to the active site of an enzyme, there are a variety of tools that enzymes use to catalyze the conversion of substrate to product (14-16).
The peptidoglycan strands are targeted by the D,D-carboxypeptidase, resulting in the hydrolysis of the terminal D-alanine residue. This prevents the cross-linking of the peptidoglycans in the cell wall and results in the eventual lysis of the cell. Chemical structures highlighting the close structural analogy of the 13-lactam antibiotics (right) to the D-Ala-D-Ala dipeptide (left).
The interaction of ~-lactamases with ~-lactam antibiotics results in the formation of an acyl-enzyme intermediate, which is rapidly hydrolyzed by the bound water molecule. Despite the wealth of information on catalysis in the 13-lactamase RTEM-1, questions remain.
Chapter 4
A third class of ~-lactamases, class C enzymes, consist of the largest enzymes and use a nucleophilic serine in the active site (8). To date, the most elusive aspect of the detailed mechanism of ~-lactamase action is the nature of the activation of the serine hydroxyl of the active site. The authors suggested that Lys 73 interacts with Ser 70, but with the electrostatic assistance of the positively charged ammonium groups of Lys 73.
However, upon reaction with ethyleneimine, the activity of the K73C mutant was restored to nearly wild-type levels ( 17 ). Both 15N and Be nuclei can be incorporated into the labeling molecule (Figures 7 and 8), and the pKa of the resulting aminoethylcysteine can be determined by an NMR titration experiment. Similarly, the Be core in the amine undergoes a −6 ppm shift upon protonation of the amine (Figure 10) (19).
Thus, the lion's share of proton signals from the enzyme is eliminated, and in the case of the above-mentioned labeling experiments, only very specific protons are visible. Using either of these experimental approaches, the pKa of the native Lys 73 amine side chain could be inferred from the experimental pKa value of the aminoethylcysteine side chain, which is −1 pKa unit lower than that of lysine ( 22 ). The formation of pyridine hydrochloride is evidenced by the presence of white crystals at the bottom of the flask.
The first is a modification of the Gabriel synthesis (26) proposed by Brown and van Gulick (27). Plasmid DNA was sequenced using a modification of the Sanger dideoxy method for denatured, double-stranded DNA ( 36 ). Kodak AR x-ray film was then exposed in the presence of the sequencing gel for 12 hours.
It contains a 13-lactamase gene under the control of the tac promoter (39) and a kanamycin gene as a selectable marker. The first involved partial denaturation of the protein by addition of solid urea to a final concentration of 4.0 M urea. The proton part of this sequence consists of a simple spin-echo experiment with the receiver phase changing from scan to scan.
RESULTS
Reduction of 13C-BOC-glycine to 13C-BOC-ethanolamine was achieved using the borane reagent BH3-THF. The synthesis of 13C-2-bromoethylamine hydrobromide was based on X modifications of the original Organic Synthesis Prep. We recorded the 13C spectrum of aminoethylcysteine (Aldrich) to determine where the 13C nuclei of the a- and [3- to amine carbons would be.
This system appeared to be ideal for the large amounts of protein required for these NMR experiments, so an attempt was made to insert the K73C 13-lactamase gene into the pET system. Unfortunately, repeated attempts at protein expression resulted in the accumulation of large amounts of the preprotein in the cytoplasm, with almost no processing and secretion into the periplasm. These two factors conspired to doom pKa determination by direct observation of the change in 15N chemical shift with pH.
Interestingly, these two conditions yielded protein showing only one non-artifact peak, but the chemical shifts of the peaks differed by -0.3 ppm. The identity of the labeled species and the reason for the differences in chemical shifts are not known. It was very encouraging that a peak rose out of the aliphatic region in the 13C-edited spectra.
Calculated chemical shifts for protons in the aminoethyl cysteine residue range from -2.8 to 3.45 ppm, depending on the protonation state of the amine. The PKa of the ionizing group adjacent to the observed protons can be estimated by plotting the chemical shift change. It is conceivable that this altered conformation of the protein could induce a decreased pK3 for Lys 73, thereby deprotonating the amine so that it would be available to act as a general base in the acylation step of the reaction.
The pKa of the K73AEC derivative could therefore be tested as free enzyme, and also in the presence of bound substrate. This will indicate whether the conformational changes affect the chemical behavior of the active site residues. The pKa of the group being ionized affecting the protons seen is -8 (Figure 39).
The K73AEC enzyme is known to have only -50% of wild-type activity. Also, further studies using 15N chemical editing experiments are planned, which should unequivocally identify the pKa of the amine functionality at position 73.
